Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a–d ) Live cell TIRF microscopy images showing ( a) FLAG–β1AR (blue) or (c) FLAG–β2AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Average enrichment at CCSs after 10 μM isoproterenol treatment for ( b) FLAG–β1AR ( d ) FLAG–β2AR (n=14 and 15 cells, respectively, from 3 independent experiments, data shown as mean ± s.e.m.). ( e ) Live cell TIRF microscopy images of HEK 293 cells co-expressing super ecliptic pHluorin–β2AR (blue), β-arrestin-2–mApple (green), and clathrin-light-chain–TagBFP (red) before and after 10 μM isoproterenol treatment. ( f ) Timelapse of individual pre-existing CCSs from panel e. Scale bars, 5 μm. ( a, c, e, f) show representative images from 3 independent experiments.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β1AR after the following treatments: 10 μM isoproterenol (green, n=14 cells), 15 minute pretreatment with 10 μM CGP 20712A and 10 μM isoproterenol treatment (red, n=12 cells), 10 μM CGP 20712A alone (gray, n=12 cells). Data shown for the 10 μM isoproterenol condition are replotted from . (b) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells transfected with the indicated receptor or empty vector and treated with 10 μM isoproterenol. (c) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated with 10 μM isoproterenol or 10 μM dobutamine (n=5 or 4 cells, respectively, from 2 independent experiments). (d) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated as indicated (n=12 cells). ( e ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β1AR (blue), β-arrestin-1–mVenus (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( f ) Enrichment into CCSs (n=7 cells from 3 independent experiments). (g) Maximum β-arrestin-1–mVenus enrichment at CCSs in HEK 293 cells transfected with FLAG-β1AR or empty vector and treated with 10 μM isoproterenol (n=7 and 11 cells from 3 independent experiments, p=0.0023 using an unpaired t test with Welch’s correction). (h) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β2AR after the following treatments: 10 μM isoproterenol (green, n=15 cells), 15 minute pretreatment with 10 μM ICI 118,551 and then 10 μM isoproterenol treatment (red, n=14 cells), 10 μM μM ICI 118,551 (gray, n=12). Data shown for the 10 μM isoproterenol condition are replotted from . ( i ) Fluorescence intensity profiles from lines shown in . ( j ) Time-dependent correlation coefficient of line scans across cells derived from immobilization experiments shown in ; n=3). ( k ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β2AR-GFP and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Fluorescence from the Alexa647 conjugated FLAG antibody shown in blue and GFP fluorescence shown in green. ( l ) Difference in GFP and Alexa647 fluorescence enrichment at CCSs in cells co-expressing FLAG-β1ARs (red), FLAG-β2ARs (blue) and β-arrestin-2-GFP or FLAG-β2AR-GFP (black). Cells were labeled with Alexa647 conjugated FLAG antibody for 10 minutes prior to live cell imaging. Data were derived from the experiments shown in (blue line, n=14 cells from 3 independent experiments), (red line, n=15 cells from 3 independent experiments), and (black line n=12 cells from 3 independent experiments). ( m ) Plot of β-arrestin/GPCR stoichiometry calculated from the data displayed in panel k, calibrated according to the doubly labeled FLAG-β2AR-GFP reference construct defining 1:1 stoichiometry (For β1AR and β2AR, n=14 and 15 cells, respectively, from 3 independent experiments). A correction index was calculated by dividing GFP fluorescence by Alexa647 (FLAG) fluorescence in CCSs. This correction index was then applied to receptor and β-arrestin-2 enrichment in CCSs to determine β-arrestin-2/GPCR stoichiometry throughout the time course. Images were captured continuously at 0.5 Hz and stoichiometry values over the time course were calculated using a rolling average with 50-frame window size. Scale bar, 5 μm. Scatter plots show overlay of mean and s.e.m. ( a, d, h, j, l) show data as mean ± s.e.m. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Microscopy, Fluorescence, Derivative Assay, Labeling, Live Cell Imaging, Construct

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–mu opioid receptor (MOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM DAMGO treatment. ( b ) Average FLAG-MOR and β-arrestin-2–GFP enrichment at CCSs after treatment with 10 μM DAMGO (n=12 cells). ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs for HEK 293 cells expressing FLAG-MOR or empty vector and treated with 10 μM DAMGO (n=12 cells per condition from 3 independent experiments; p=<0.0001 using a two-tailed unpaired t test with Welch’s correction). ( d ) Live cell TIRF microscopy images showing FLAG–kappa opioid receptor (KOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM dynorphin treatment. ( e ) Enrichment into CCSs after bath application of 10 μM dynorphin (n=18 cells). ( f ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells expressing FLAG-KOR or empty vector and treated with 10 μM dynorphin (n=18, 13 cells, respectively, from 3 independent experiments; p=0.0028 using a two-tailed unpaired t test with Welch’s correction). ( g ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red), before and after 10 μM quinpirole treatment. ( h ) Enrichment into CCSs after bath application of 10 μM quinpirole (n=12 cells). ( i ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-DRD2 or untransfected and treated with 10 μM quinpirole (n=11, 12 cells from 3 independent experiments; p=0.0095 using a two-tailed unpaired t test with Welch’s correction). ( a, d, g ) show representative images from 3 independent experiments. ( b, e, h ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–β1AR truncated at the 415 th amino acid (415T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs after 10 μM isoproterenol for cells co-expressing the indicated FLAG–β1AR receptor (n=10, 12 cells, respectively, from 3 independent experiments, p=0.5825 calculated using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–β2AR truncated at the 365 th amino acid (365T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells treated with 10 μM isoproterenol and either transfected with FLAG-β2AR or empty vector (n=11, 13 cells, respectively, from 3 independent experiments, p=0.0269 calculated using a two-tailed unpaired t test with Welch’s correction). ( e ) Maximum β-arrestin-2–GFP enrichment at CCSs for cells co-expressing the indicated FLAG–β2AR receptor and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments, p=0.0606 calculated using a two-tailed unpaired t test). ( f ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( g ) Initial enrichment in CCSs before 10 μM quinpirole treatment and ( h ) maximum enrichment after 10 μM quinpirole treatment (n=12 cells from 3 independent experiments; p=0.19 and 0.4873, respectively, using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β1AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 5 μM forskolin (fsk) treatment. ( j ) Initial enrichment in CCSs before 5 μM forskolin (fsk) treatment and ( k ) maximum enrichment after 5 μM fsk treatment (n=12 cells from 3 independent experiments; p=0.6325 and 0.0971, respectively, using a two-tailed unpaired t test). ( l ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2–GFP KNC mutant (green) and clathrin-light-chain–DsRed (red before and after 10 μM isoproterenol treatment. ( m ) Initial enrichment in CCSs before 10 μM isoproterenol treatment and ( n ) maximum enrichment after 10 μM isoproterenol (n=9 (WT) or 8 (KNC) cells from 3 independent experiments; p=0.6681( m ) and p=0.001 ( n ) using a two-tailed unpaired t test with Welch’s correction). ( a, c, f, I, l ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05, ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Mutagenesis

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images of COS-1 cells co-expressing FLAG–β2AR truncated at the 341 st amino acid (341T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells treated with 10 μM isoproterenol and co-expressing the indicated FLAG–β2AR (n=11 cells from 3 independent experiments, p=0.5634 using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–DRD2 G protein biased mutant (G prot, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( d ) Average (data shown as mean ± s.e.m.) and ( e ) maximum enrichment of β-arrestin-2–GFP into CCSs in cells expressing wild-type (green) or G protein biased mutant versions (gray) of FLAG-DRD2 and treated with 10 μM quinpirole (n=11 (WT) and 14 (G prot) cells from 3 independent experiments, p=0.013 using a two-tailed unpaired t test using Welch’s correction). ( a ) and ( c) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Two Tailed Test, Mutagenesis

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR (blue), clathrin-light-chain–DsRed (red), and wild-type (top, green) or finger loop proximal mutant (bottom, green) β-arrestin-2–GFP without agonist treatment. Clustering index measuring constitutive activation of the indicated ( b ) β-arrestin-2–GFP or ( c ) β-arrestin-1–mVenus constructs without agonist treatment (n=12 cells from 3 independent experiments, p<0.0001 and 0.0008, respectively, using a two-tailed unpaired t test). ( d ) Snapshot from molecular dynamics simulations of inactive-state β-arrestin-1 in which K77 and E313 occasionally form a stable salt bridge. This salt bridge formed 6% of the time in inactive-state simulations (six simulations totaling 26.7 μs); it may form more frequently on longer timescales. It formed in only a few frames of active-state simulations (0.2% of the time across six simulations totaling 29.3 μs in length). ( e ) Clustering index of the indicated β-arrestin-2–GFP construct without agonist treatment. Statistics were calculated using a two-tailed unpaired t test (for K78E, n=12 cells from 3 independent experiments, p=0.0003; for E314K, n=12 cells from 3 independent experiments, p<0.0001. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. *** p < 0.001.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Mutagenesis, Activation Assay, Construct, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and the polar core mutant of β-arrestin-2–GFP (green) in the absence of agonist treatment. ( b ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonist treatment. Statistical significance was calculated using an two-tailed unpaired t test with Welch’s correction (polar core mutant: n=12 cells from 3 independent experiments, p<0.0001; finger loop proximal mutant: n=16 cells from 3 independent experiments p<0.0001; R77A: n=12 cells from 3 independent experiments, p=0.0403; K78A: n=12 cells from 3 independent experiments, p=0.0016). WT and finger loop proximal mutant data replotted from . ( c ) Association of β-arrestin-2–GFP constructs with the adaptin beta subunit of AP-2 in the absence of agonist treatment. Molecular mass markers (in kDa) are shown on the right side of blots. For gel source data, see . The representative Western blots in panel c are representative of 3 independent experiments, quantified in ( d ), and shown as AP-2/GFP intensity in the immunoprecipitation conditions (n=3 independent experiments, p=0.0218 using a two-tailed unpaired t test). ( e ) Measurement of β-arrestin-2–GFP construct expression in cell lysates from panel c. ( f–i ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and β-arrestin-2–GFP with the indicated point mutations (green) in the absence of agonist treatment. Detailed description of β-arrestin mutations are provided in . ( a, f–i ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Mutagenesis, Construct, Two Tailed Test, Western Blot, Immunoprecipitation, Expressing

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: Graphical representation of β-arrestin interaction domains without ( a ) and with ( b ) βAR activation by isoproterenol. ( c ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP lipid mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( e ) Representative western blot (from 4 independent experiments) of purified wild-type and lipid mutant versions of β-arrestin-1(1-393) immunoprecipitation with PIP2-coated agarose beads and quantified in ( f ) as percent of input protein (n=4 independent experiments, p=0.0142 using a two-tailed unpaired t test). For gel source data, see . ( g ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (F191G, L192G) lipid anchor mutant mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( h ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing the indicated β-arrestin-2–GFP construct and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments; p=0.9227 calculated using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. ( j ) Representative images of HEK 293 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP lipid and CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. Representative β-arrestin images false colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (data shown as mean ± s.e.m.), respectively, from cells co-expressing FLAG–β1ARs (n=12 cells per condition) without isoproterenol treatment ( k–m ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( n–p ), lipid mutant ( q–s ), CCS mutant ( t–v ), and CCS and lipid mutant ( w–y ). Wild-type β-arrestin-2–GFP maximum enrichment at CCSs shown in panels r, u, x is replotted from panel o. Live cell TIRF microscopy images showing cells before and after 10 μM isoproterenol treatment and co-expressing FLAG–β1AR (blue), clathrin-light-chain–DsRed (red), and the following GFP labeled versions of β-arrestin-2 (green): ( z ) wild-type, ( aa ) lipid mutant, and ( ab ) CCS mutant, and ( ac ) CCS and lipid mutant. ( ad ) Live cell TIRF microscopy images showing FLAG-β2AR and the indicated β-arrestin-2-GFP construct in the absence of agonist treatment. ( ae ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonists treatment. Detailed description of β-arrestin mutations are provided in . ( c, d, g, i, j, k, n, q, t, w, z, aa, ab, ac, ad ) show representative images from 3 independent experiments. For ( r, u, x) n=12 cells from 3 independent experiments; statistical significance was calculated using an unpaired t test with Welch’s correction, p=0.0007, 0.0018, and 0.0012, respectively. For ( ae ), statistical significance was calculated using an unpaired t test with Welch’s correction, n=12 (WT) and 16 (finger loop proximal mutant) from 3 independent experiments, p<0.0001; n=12 (WT) and 15 (finger loop proximal & lipid mutant) from 3 independent experiments, p=0.5464). WT and finger loop proximal mutant data replotted from . Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Activation Assay, Microscopy, Mutagenesis, Western Blot, Purification, Immunoprecipitation, Two Tailed Test, Expressing, Construct, Fluorescence, Labeling

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR, the indicated β-arrestin-2–GFP construct, and clathrin-light-chain–DsRed. Shown are β-arrestin images false-colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (shown as mean ± s.e.m), respectively, from cells co-expressing FLAG–β2ARs without isoproterenol treatment ( a–c ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( d–f ), lipid mutant ( g–i ), CCS mutant ( j–l ), and CCS and lipid mutant ( m–o ); n=12 cells per condition. Wild-type β-arrestin-2–GFP maximum enrichment in panel h is replotted from panel e and panel n is replotted from panel k. Statistics were calculated using a two-tailed unpaired t test with Welch’s correction. For ( h ) n=12 and 11 cells, respectively, from 3 independent experiments and p=0.0006. For ( k ) n=10 cells from 3 independent experiments and p=0.0102. For ( n ) n=10 cells from 3 independent experiments and p=0.0022. provides detailed description of β-arrestin mutations. Scatter plots show overlay of mean and s.e.m. scale bars, 5 μm. ** p < 0.01; *** p < 0.001.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Construct, Fluorescence, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell microscopy images of HEK 293 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol treatment. ( b ) Normalized plasma membrane (PM) fluorescence of β-arrestin-2–GFP lipid mutant in cells co-expressing FLAG–β2AR-V2R (n=12 cells from 3 independent experiments) when treated with 10 μM isoproterenol. ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing indicated β-arrestin-2–GFP construct before and after activation of FLAG-β2AR-V2R C tail with 10 μM isoproterenol (n=10, 12 cells, respectively, from 3 independent experiments; p=0.6433 using a two-tailed unpaired t test). ( d ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) or vehicle (DMSO) before 10 μM isoproterenol treatment. ( e ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( f ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP (green), and CLC-dsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before before 10 μM isoproterenol treatment. ( g ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR or FLAG–β2AR-V2R when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( a, d, f ) show representative images from 3 independent experiments. ( b, e, g ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Mutagenesis, Fluorescence, Construct, Activation Assay, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a–d ) Representative images (from at least 3 independent experiments) of ( a ) photoactivatable (PA) mCherry-β1AR (green) or ( b ) PAmCherry-β2AR (green) trajectories and ( c ) PAmCherry-β-arrestin-2 trajectories (green) with β1AR expression or ( d ) PAmCherry-β-arrestin-2 (green) trajectories with β2AR expression from sptPALM analysis overlaid with a clathrin marker (red) after 10 μM isoproterenol treatment. ( e ) False positive corrected diffusion coefficients (D) of PAmCherry-β2AR, β-arrestin-2-PAmCherry, and PAmCherry-PLCδ1-PH in live cells after 10 μM isoproterenol treatment (n=13, 21, and 8 cells, respectively). β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. False positive corrected distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with ( f) FLAG-β1AR (n=13 and 17 cells, respectively, from 3 independent experiments; statistical significance of the immobile fractions was calculated using a two-tailed unpaired t test, p<0.0001) or ( g ) FLAG-β2AR (n=21 and 10 cells, respectively, from 3 independent experiments; statistical significance of the immobile fractions was calculated using a two-tailed unpaired t test, p=0.002) β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel e. ( h ) COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) were treated with 10 μM isoproterenol for 3 minutes before β-arrestin-2-GFP photobleaching. Shown are representative images (from 3 independent experiments) of the photobleached area. β-arrestin-2 clustering index over the course of the photobleaching experiment in cells co-expressing activated ( i ) FLAG-β1AR (n=12 cells from 3 independent experiments) or ( j ) FLAG-β2AR (n=15 and 13 cells for unbleached and photobleached conditions, respectively, from 3 independent experiments). Data are shown as mean ± s.e.m. Scale bars, 0.5 μm.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Expressing, Marker, Diffusion-based Assay, Mutagenesis, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: (a) Representative image of a clathrin mask (green) generated from a CLC-GFP image (red). Representative diffusion maps overlaid with the clathrin mask for HEK 293 cells and treated with 10 μM isoproterenol expressing ( b ) PAmCherry-β1AR, ( c ) PAmCherry-β2AR, ( d ) β-arrestin-2-PAmCherry coexpressed with FLAG-β1AR, ( e ) β-arrestin-2-PAmCherry coexpressed with FLAG-β2AR ( f ) Distribution of diffusion coefficients (D) of false positive detections from HEK 293 cells expressing FLAG-β2AR and imaged under standard sptPALM acquisition conditions to determine contribution of false positive detections in the experimental setup and analysis. ( g ) Distribution of diffusion coefficients (D) of PAmCherry-β2AR, PAmCherry-PLCδ1-PH, and β-arrestin-2-PAmCherry in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13, 21, and 8 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. ( h ) Average MSD plots derived from sptPALM analysis of PAmCherry-β1AR and PAmCherry-β2AR trajectories in HEK 293 cells treated with 10 μM isoproterenol (n=8 and 13 cells, respectively). ( i ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β1AR in live HEK 293 cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13 and 17 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. ( j ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in cells co-expressing FLAG-β1AR and treated with 10 μM isoproterenol (n=13 and 17 cells, respectively). ( k ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β2AR in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=21 and 10 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel d. ( l ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in HEK 293 cells co-expressing FLAG-β2AR and treated with 10 μM isoproterenol (n=21 and 10 cells, respectively). ( m ) Immobile and ( n) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β1AR after 10 μM isoproterenol treatment. ( o ) Immobile and ( p) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β2AR after 10 μM isoproterenol treatment. Trajectory detections are false colored based on the density of detections at each pixel. Error bars represent s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images. ( q ) Proposed cellular pathway for catalytic activation of β-arrestin. ( r ) Representative microscopy images of COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) that were treated with 10 μM isoproterenol for 3 minutes. Then, β-arrestin-2-GFP was photobleached in the indicated yellow region (shown in inset; insets are also shown in ). ( a, b, c, d, e, m, n, o, p, and r ) show representative examples from at least 3 independent experiments. ( f–l ) show data as mean ± s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images; 5 μm for FRAP larger images and 0.5 μm for the insets.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Generated, Diffusion-based Assay, Expressing, Derivative Assay, Mutagenesis, Marker, Activation Assay, Microscopy

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Schematic depicting the proposed co-existence of catalytic and scaffolding mechanisms of β-arrestin trafficking tuned according to tail binding affinity, emphasizing the difference in tail versus core interactions (shaded boxes). The tail interaction, requiring GPCR phosphorylation (Rp) drives the scaffold mechanism through its essential role in stable GPCR/β-arrestin complex formation. The core interaction mediates catalysis by providing a kinetically favorable path for β-arrestin to remain captured at the PM irrespective of GPCR dissociation. Such capture requires phosphoinositide binding to the β-arrestin C-domain, explaining why the phosphoinositide requirement is specific to the catalytic mechanism and can be overcome by formation of a sufficiently sufficient stable scaffold complex requiring the phosphorylated GPCR tail. Primary energy inputs maintaining each proposed trafficking cycle are indicated by red arrows. The present results identify a specific requirement of the catalytic mechanism for phosphoinositide binding to the C-domain but they do not exclude binding also in the scaffold complex (which we think is likely). We also cannot presently rule out the possible existence of additional interaction(s) in the catalytic mechanism, such as phosphoinositide binding also to the β-arrestin N-domain that has the potential to displace the β-arrestin C-terminus . ( b ) Representative images (from 3 independent experiments) before and after 10 μM isoproterenol treatment of cells expressing chimeric FLAG-tagged β1AR-V2Rs and imaged live with TIRF microscopy. Profiles of FLAG-β2AR and β-arrestin-2–GFP average enrichment into CCSs in COS-1 cells expressing either an empty vector construct ( c ) or GRK2 ( d ) and treated with 10 μM isoproterenol (n=15 or 12 cells, respectively, from 3 independent experiments). ( e ) Difference in enrichment values between β-arrestin-2–GFP and β2AR from panels c and d showing the effect of GRK2 overexpression. ( f ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β1AR and exposed to 10 μM isoproterenol for the indicated time period. ( g ) Quantification of ERK1/2 activation from the western blots in panel a (n=5 independent experiments, p=0.004 using a one-way ANOVA). ( h ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β2AR and exposed to 10 μM isoproterenol for the indicated time period. ( i ) Quantification of ERK1/2 activation from the western blots in panel c (n=5 independent experiments). ( f ) and ( h ) show representative Western blots from 5 independent experiments. Data shown as mean ± s.e.m. For gel source data, see . Error bars represent s.e.m. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Scaffolding, Binding Assay, Expressing, Microscopy, Plasmid Preparation, Construct, Over Expression, Western Blot, Knock-Out, CRISPR, Activation Assay

Journal:

Article Title: Independent ?-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

doi: 10.1073/pnas.1834556100

Figure Lengend Snippet: [Sar1,Ile4,Ile8]Ang II-bound AT1A receptors and Ang II-bound DRY/AAY mutant receptors are able to induce translocation of β-arrestin 2-GFP into endocytic vesicles. HEK-293 cells were transiently transfected with expression vectors encoding β-arrestin 2-GFP and wild-type (B and D) or DRY/AAY mutant AT1A receptors (A and C). Cells were not stimulated (A), stimulated with 160 nM Ang II (B and C), or stimulated with 30 μM [Sar1,Ile4,Ile8]Ang II (D) for 30 min. Cells were fixed with 5% formaldehyde, and the recruitment of β-arrestin 2-GFP was examined by confocal microscopy. The distribution pattern of β-arrestin 2-GFP in cells that were not stimulated is the same between cells expressing the wild-type and DRY/AAY mutant AT1A receptors. The results shown are representative of three experiments.

Article Snippet: GFP-tagged β-arrestin 2 (β-arrestin 2-GFP) was made by cloning rat β-arrestin 2 cDNA in frame in pEGFPN1 (Clontech) between the Hin dIII and Apa I sites.

Techniques: Mutagenesis, Translocation Assay, Transfection, Expressing, Confocal Microscopy

Journal:

Article Title: Independent ?-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

doi: 10.1073/pnas.1834556100

Figure Lengend Snippet: β-Arrestin 2 mediates ERK1/2 activation induced by [Sar1,Ile4,Ile8]Ang II-bound AT1A receptors and the Ang II-bound DRY/AAY mutant receptors. (A) HEK-293 cells were transfected with control siRNA and expression vectors encoding wild-type or DRY/AAY-mutant AT1A receptors. Three days after transfection, cells were cultured in serum-free medium for 4 h and stimulated with 160 nM Ang II or 30 μM [Sar1,Ile4,Ile8]Ang II for 5 min. The activation of ERK1/2 was determined by immunoblotting with a phospho-ERK1/2-specific antibody. The levels of phospho-ERK1/2 were then quantified and normalized to the phospho-ERK1/2 signal induced by Ang II-stimulated wild-type AT1A receptors in each experiment. **, P < 0.01, compared with the phospho-ERK1/2 induced by Ang II-stimulated wild-type AT1A receptors (n = 4). (B–G) HEK-293 cells were transfected with control siRNA (CTL siRNA) or β-arrestin 2 siRNA and expression vectors encoding wild-type (WT) (B–E) or DRY/AAY-mutant (F and G)AT1A receptors. The activation of ERK1/2 in cells stimulated with different concentrations of Ang II (B, C, F, and G) or [Sar1,Ile4,Ile8]Ang II (D and E) was determined as described in A. The amounts of total ERK1/2 and β-arrestin 2 were determined by stripping the membrane and immunoblotting for total ERK1/2 and β-arrestin 2. The effect of β-arrestin 2 siRNA on dose-dependent ERK1/2 activation induced by Ang II (C, n = 4)- or [Sar1,Ile4,Ile8]Ang II-stimulated wild-type AT1A receptors (E, n = 6), or the Ang II-stimulated DRY/AAY mutant receptors (G, n = 4) was plotted by normalizing to the maximum phospho-ERK1/2 signal in each experiment. *, P < 0.05; **, P < 0.01, compared with the phospho-ERK1/2 signal in the corresponding CTL siRNA-transfected cells.

Article Snippet: GFP-tagged β-arrestin 2 (β-arrestin 2-GFP) was made by cloning rat β-arrestin 2 cDNA in frame in pEGFPN1 (Clontech) between the Hin dIII and Apa I sites.

Techniques: Activation Assay, Mutagenesis, Transfection, Expressing, Cell Culture, Western Blot, Stripping Membranes

Journal:

Article Title: Independent ?-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

doi: 10.1073/pnas.1834556100

Figure Lengend Snippet: Effect of the PKC inhibitor Ro-31-8425 on Ang II- and [Sar1,Ile4,Ile8]Ang II (SII)-induced ERK1/2 activation. (A) HEK-293 cells were transfected with control (CTL) siRNA or β-arrestin 2 siRNA, and expression vectors encoding wild-type AT1A receptors. Cells were pretreated with or without Ro-31-8425 for 10 min followed by 5-min stimulation by Ang II or [Sar1,Ile4,Ile8]Ang II. NS, no stimulation. As a control for the effectiveness of the Ro-31-8425, HEK-293 cells were pretreated with or without Ro-31-8425 for 10 min followed by 5-min stimulation by PMA. The activation of ERK1/2 was determined by immunoblotting with a phospho-ERK1/2-specific antibody (p-Erk1/2). (B) The effects of β-arrestin 2 siRNA and Ro-31-8425 on Ang II-induced ERK1/2 activation were compared by normalizing each phospho-ERK1/2 signal to the response induced by Ang II in non-inhibitor-treated cells transfected with control siRNA (n = 4). *, P < 0.01, compared with the ERK1/2 activation induced by Ang II-stimulated wild-type AT1A receptors; **, P < 0.01, compared as indicated.

Article Snippet: GFP-tagged β-arrestin 2 (β-arrestin 2-GFP) was made by cloning rat β-arrestin 2 cDNA in frame in pEGFPN1 (Clontech) between the Hin dIII and Apa I sites.

Techniques: Activation Assay, Transfection, Expressing, Western Blot

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a–d ) Live cell TIRF microscopy images showing ( a) FLAG–β1AR (blue) or (c) FLAG–β2AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Average enrichment at CCSs after 10 μM isoproterenol treatment for ( b) FLAG–β1AR ( d ) FLAG–β2AR (n=14 and 15 cells, respectively, from 3 independent experiments, data shown as mean ± s.e.m.). ( e ) Live cell TIRF microscopy images of HEK 293 cells co-expressing super ecliptic pHluorin–β2AR (blue), β-arrestin-2–mApple (green), and clathrin-light-chain–TagBFP (red) before and after 10 μM isoproterenol treatment. ( f ) Timelapse of individual pre-existing CCSs from panel e. Scale bars, 5 μm. ( a, c, e, f) show representative images from 3 independent experiments.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β1AR after the following treatments: 10 μM isoproterenol (green, n=14 cells), 15 minute pretreatment with 10 μM CGP 20712A and 10 μM isoproterenol treatment (red, n=12 cells), 10 μM CGP 20712A alone (gray, n=12 cells). Data shown for the 10 μM isoproterenol condition are replotted from . (b) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells transfected with the indicated receptor or empty vector and treated with 10 μM isoproterenol. (c) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated with 10 μM isoproterenol or 10 μM dobutamine (n=5 or 4 cells, respectively, from 2 independent experiments). (d) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated as indicated (n=12 cells). ( e ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β1AR (blue), β-arrestin-1–mVenus (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( f ) Enrichment into CCSs (n=7 cells from 3 independent experiments). (g) Maximum β-arrestin-1–mVenus enrichment at CCSs in HEK 293 cells transfected with FLAG-β1AR or empty vector and treated with 10 μM isoproterenol (n=7 and 11 cells from 3 independent experiments, p=0.0023 using an unpaired t test with Welch’s correction). (h) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β2AR after the following treatments: 10 μM isoproterenol (green, n=15 cells), 15 minute pretreatment with 10 μM ICI 118,551 and then 10 μM isoproterenol treatment (red, n=14 cells), 10 μM μM ICI 118,551 (gray, n=12). Data shown for the 10 μM isoproterenol condition are replotted from . ( i ) Fluorescence intensity profiles from lines shown in . ( j ) Time-dependent correlation coefficient of line scans across cells derived from immobilization experiments shown in ; n=3). ( k ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β2AR-GFP and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Fluorescence from the Alexa647 conjugated FLAG antibody shown in blue and GFP fluorescence shown in green. ( l ) Difference in GFP and Alexa647 fluorescence enrichment at CCSs in cells co-expressing FLAG-β1ARs (red), FLAG-β2ARs (blue) and β-arrestin-2-GFP or FLAG-β2AR-GFP (black). Cells were labeled with Alexa647 conjugated FLAG antibody for 10 minutes prior to live cell imaging. Data were derived from the experiments shown in (blue line, n=14 cells from 3 independent experiments), (red line, n=15 cells from 3 independent experiments), and (black line n=12 cells from 3 independent experiments). ( m ) Plot of β-arrestin/GPCR stoichiometry calculated from the data displayed in panel k, calibrated according to the doubly labeled FLAG-β2AR-GFP reference construct defining 1:1 stoichiometry (For β1AR and β2AR, n=14 and 15 cells, respectively, from 3 independent experiments). A correction index was calculated by dividing GFP fluorescence by Alexa647 (FLAG) fluorescence in CCSs. This correction index was then applied to receptor and β-arrestin-2 enrichment in CCSs to determine β-arrestin-2/GPCR stoichiometry throughout the time course. Images were captured continuously at 0.5 Hz and stoichiometry values over the time course were calculated using a rolling average with 50-frame window size. Scale bar, 5 μm. Scatter plots show overlay of mean and s.e.m. ( a, d, h, j, l) show data as mean ± s.e.m. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Microscopy, Fluorescence, Derivative Assay, Labeling, Live Cell Imaging, Construct

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–mu opioid receptor (MOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM DAMGO treatment. ( b ) Average FLAG-MOR and β-arrestin-2–GFP enrichment at CCSs after treatment with 10 μM DAMGO (n=12 cells). ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs for HEK 293 cells expressing FLAG-MOR or empty vector and treated with 10 μM DAMGO (n=12 cells per condition from 3 independent experiments; p=<0.0001 using a two-tailed unpaired t test with Welch’s correction). ( d ) Live cell TIRF microscopy images showing FLAG–kappa opioid receptor (KOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM dynorphin treatment. ( e ) Enrichment into CCSs after bath application of 10 μM dynorphin (n=18 cells). ( f ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells expressing FLAG-KOR or empty vector and treated with 10 μM dynorphin (n=18, 13 cells, respectively, from 3 independent experiments; p=0.0028 using a two-tailed unpaired t test with Welch’s correction). ( g ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red), before and after 10 μM quinpirole treatment. ( h ) Enrichment into CCSs after bath application of 10 μM quinpirole (n=12 cells). ( i ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-DRD2 or untransfected and treated with 10 μM quinpirole (n=11, 12 cells from 3 independent experiments; p=0.0095 using a two-tailed unpaired t test with Welch’s correction). ( a, d, g ) show representative images from 3 independent experiments. ( b, e, h ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–β1AR truncated at the 415 th amino acid (415T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs after 10 μM isoproterenol for cells co-expressing the indicated FLAG–β1AR receptor (n=10, 12 cells, respectively, from 3 independent experiments, p=0.5825 calculated using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–β2AR truncated at the 365 th amino acid (365T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells treated with 10 μM isoproterenol and either transfected with FLAG-β2AR or empty vector (n=11, 13 cells, respectively, from 3 independent experiments, p=0.0269 calculated using a two-tailed unpaired t test with Welch’s correction). ( e ) Maximum β-arrestin-2–GFP enrichment at CCSs for cells co-expressing the indicated FLAG–β2AR receptor and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments, p=0.0606 calculated using a two-tailed unpaired t test). ( f ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( g ) Initial enrichment in CCSs before 10 μM quinpirole treatment and ( h ) maximum enrichment after 10 μM quinpirole treatment (n=12 cells from 3 independent experiments; p=0.19 and 0.4873, respectively, using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β1AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 5 μM forskolin (fsk) treatment. ( j ) Initial enrichment in CCSs before 5 μM forskolin (fsk) treatment and ( k ) maximum enrichment after 5 μM fsk treatment (n=12 cells from 3 independent experiments; p=0.6325 and 0.0971, respectively, using a two-tailed unpaired t test). ( l ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2–GFP KNC mutant (green) and clathrin-light-chain–DsRed (red before and after 10 μM isoproterenol treatment. ( m ) Initial enrichment in CCSs before 10 μM isoproterenol treatment and ( n ) maximum enrichment after 10 μM isoproterenol (n=9 (WT) or 8 (KNC) cells from 3 independent experiments; p=0.6681( m ) and p=0.001 ( n ) using a two-tailed unpaired t test with Welch’s correction). ( a, c, f, I, l ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05, ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Mutagenesis

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images of COS-1 cells co-expressing FLAG–β2AR truncated at the 341 st amino acid (341T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells treated with 10 μM isoproterenol and co-expressing the indicated FLAG–β2AR (n=11 cells from 3 independent experiments, p=0.5634 using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–DRD2 G protein biased mutant (G prot, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( d ) Average (data shown as mean ± s.e.m.) and ( e ) maximum enrichment of β-arrestin-2–GFP into CCSs in cells expressing wild-type (green) or G protein biased mutant versions (gray) of FLAG-DRD2 and treated with 10 μM quinpirole (n=11 (WT) and 14 (G prot) cells from 3 independent experiments, p=0.013 using a two-tailed unpaired t test using Welch’s correction). ( a ) and ( c) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Two Tailed Test, Mutagenesis

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR (blue), clathrin-light-chain–DsRed (red), and wild-type (top, green) or finger loop proximal mutant (bottom, green) β-arrestin-2–GFP without agonist treatment. Clustering index measuring constitutive activation of the indicated ( b ) β-arrestin-2–GFP or ( c ) β-arrestin-1–mVenus constructs without agonist treatment (n=12 cells from 3 independent experiments, p<0.0001 and 0.0008, respectively, using a two-tailed unpaired t test). ( d ) Snapshot from molecular dynamics simulations of inactive-state β-arrestin-1 in which K77 and E313 occasionally form a stable salt bridge. This salt bridge formed 6% of the time in inactive-state simulations (six simulations totaling 26.7 μs); it may form more frequently on longer timescales. It formed in only a few frames of active-state simulations (0.2% of the time across six simulations totaling 29.3 μs in length). ( e ) Clustering index of the indicated β-arrestin-2–GFP construct without agonist treatment. Statistics were calculated using a two-tailed unpaired t test (for K78E, n=12 cells from 3 independent experiments, p=0.0003; for E314K, n=12 cells from 3 independent experiments, p<0.0001. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. *** p < 0.001.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Mutagenesis, Activation Assay, Construct, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and the polar core mutant of β-arrestin-2–GFP (green) in the absence of agonist treatment. ( b ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonist treatment. Statistical significance was calculated using an two-tailed unpaired t test with Welch’s correction (polar core mutant: n=12 cells from 3 independent experiments, p<0.0001; finger loop proximal mutant: n=16 cells from 3 independent experiments p<0.0001; R77A: n=12 cells from 3 independent experiments, p=0.0403; K78A: n=12 cells from 3 independent experiments, p=0.0016). WT and finger loop proximal mutant data replotted from . ( c ) Association of β-arrestin-2–GFP constructs with the adaptin beta subunit of AP-2 in the absence of agonist treatment. Molecular mass markers (in kDa) are shown on the right side of blots. For gel source data, see . The representative Western blots in panel c are representative of 3 independent experiments, quantified in ( d ), and shown as AP-2/GFP intensity in the immunoprecipitation conditions (n=3 independent experiments, p=0.0218 using a two-tailed unpaired t test). ( e ) Measurement of β-arrestin-2–GFP construct expression in cell lysates from panel c. ( f–i ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and β-arrestin-2–GFP with the indicated point mutations (green) in the absence of agonist treatment. Detailed description of β-arrestin mutations are provided in . ( a, f–i ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Mutagenesis, Construct, Two Tailed Test, Western Blot, Immunoprecipitation, Expressing

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: Graphical representation of β-arrestin interaction domains without ( a ) and with ( b ) βAR activation by isoproterenol. ( c ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP lipid mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( e ) Representative western blot (from 4 independent experiments) of purified wild-type and lipid mutant versions of β-arrestin-1(1-393) immunoprecipitation with PIP2-coated agarose beads and quantified in ( f ) as percent of input protein (n=4 independent experiments, p=0.0142 using a two-tailed unpaired t test). For gel source data, see . ( g ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (F191G, L192G) lipid anchor mutant mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( h ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing the indicated β-arrestin-2–GFP construct and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments; p=0.9227 calculated using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. ( j ) Representative images of HEK 293 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP lipid and CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. Representative β-arrestin images false colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (data shown as mean ± s.e.m.), respectively, from cells co-expressing FLAG–β1ARs (n=12 cells per condition) without isoproterenol treatment ( k–m ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( n–p ), lipid mutant ( q–s ), CCS mutant ( t–v ), and CCS and lipid mutant ( w–y ). Wild-type β-arrestin-2–GFP maximum enrichment at CCSs shown in panels r, u, x is replotted from panel o. Live cell TIRF microscopy images showing cells before and after 10 μM isoproterenol treatment and co-expressing FLAG–β1AR (blue), clathrin-light-chain–DsRed (red), and the following GFP labeled versions of β-arrestin-2 (green): ( z ) wild-type, ( aa ) lipid mutant, and ( ab ) CCS mutant, and ( ac ) CCS and lipid mutant. ( ad ) Live cell TIRF microscopy images showing FLAG-β2AR and the indicated β-arrestin-2-GFP construct in the absence of agonist treatment. ( ae ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonists treatment. Detailed description of β-arrestin mutations are provided in . ( c, d, g, i, j, k, n, q, t, w, z, aa, ab, ac, ad ) show representative images from 3 independent experiments. For ( r, u, x) n=12 cells from 3 independent experiments; statistical significance was calculated using an unpaired t test with Welch’s correction, p=0.0007, 0.0018, and 0.0012, respectively. For ( ae ), statistical significance was calculated using an unpaired t test with Welch’s correction, n=12 (WT) and 16 (finger loop proximal mutant) from 3 independent experiments, p<0.0001; n=12 (WT) and 15 (finger loop proximal & lipid mutant) from 3 independent experiments, p=0.5464). WT and finger loop proximal mutant data replotted from . Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Activation Assay, Microscopy, Mutagenesis, Western Blot, Purification, Immunoprecipitation, Two Tailed Test, Expressing, Construct, Fluorescence, Labeling

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR, the indicated β-arrestin-2–GFP construct, and clathrin-light-chain–DsRed. Shown are β-arrestin images false-colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (shown as mean ± s.e.m), respectively, from cells co-expressing FLAG–β2ARs without isoproterenol treatment ( a–c ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( d–f ), lipid mutant ( g–i ), CCS mutant ( j–l ), and CCS and lipid mutant ( m–o ); n=12 cells per condition. Wild-type β-arrestin-2–GFP maximum enrichment in panel h is replotted from panel e and panel n is replotted from panel k. Statistics were calculated using a two-tailed unpaired t test with Welch’s correction. For ( h ) n=12 and 11 cells, respectively, from 3 independent experiments and p=0.0006. For ( k ) n=10 cells from 3 independent experiments and p=0.0102. For ( n ) n=10 cells from 3 independent experiments and p=0.0022. provides detailed description of β-arrestin mutations. Scatter plots show overlay of mean and s.e.m. scale bars, 5 μm. ** p < 0.01; *** p < 0.001.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Construct, Fluorescence, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Live cell microscopy images of HEK 293 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol treatment. ( b ) Normalized plasma membrane (PM) fluorescence of β-arrestin-2–GFP lipid mutant in cells co-expressing FLAG–β2AR-V2R (n=12 cells from 3 independent experiments) when treated with 10 μM isoproterenol. ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing indicated β-arrestin-2–GFP construct before and after activation of FLAG-β2AR-V2R C tail with 10 μM isoproterenol (n=10, 12 cells, respectively, from 3 independent experiments; p=0.6433 using a two-tailed unpaired t test). ( d ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) or vehicle (DMSO) before 10 μM isoproterenol treatment. ( e ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( f ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP (green), and CLC-dsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before before 10 μM isoproterenol treatment. ( g ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR or FLAG–β2AR-V2R when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( a, d, f ) show representative images from 3 independent experiments. ( b, e, g ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Microscopy, Expressing, Mutagenesis, Fluorescence, Construct, Activation Assay, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a–d ) Representative images (from at least 3 independent experiments) of ( a ) photoactivatable (PA) mCherry-β1AR (green) or ( b ) PAmCherry-β2AR (green) trajectories and ( c ) PAmCherry-β-arrestin-2 trajectories (green) with β1AR expression or ( d ) PAmCherry-β-arrestin-2 (green) trajectories with β2AR expression from sptPALM analysis overlaid with a clathrin marker (red) after 10 μM isoproterenol treatment. ( e ) False positive corrected diffusion coefficients (D) of PAmCherry-β2AR, β-arrestin-2-PAmCherry, and PAmCherry-PLCδ1-PH in live cells after 10 μM isoproterenol treatment (n=13, 21, and 8 cells, respectively). β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. False positive corrected distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with ( f) FLAG-β1AR (n=13 and 17 cells, respectively, from 3 independent experiments; statistical significance of the immobile fractions was calculated using a two-tailed unpaired t test, p<0.0001) or ( g ) FLAG-β2AR (n=21 and 10 cells, respectively, from 3 independent experiments; statistical significance of the immobile fractions was calculated using a two-tailed unpaired t test, p=0.002) β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel e. ( h ) COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) were treated with 10 μM isoproterenol for 3 minutes before β-arrestin-2-GFP photobleaching. Shown are representative images (from 3 independent experiments) of the photobleached area. β-arrestin-2 clustering index over the course of the photobleaching experiment in cells co-expressing activated ( i ) FLAG-β1AR (n=12 cells from 3 independent experiments) or ( j ) FLAG-β2AR (n=15 and 13 cells for unbleached and photobleached conditions, respectively, from 3 independent experiments). Data are shown as mean ± s.e.m. Scale bars, 0.5 μm.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Expressing, Marker, Diffusion-based Assay, Mutagenesis, Two Tailed Test

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: (a) Representative image of a clathrin mask (green) generated from a CLC-GFP image (red). Representative diffusion maps overlaid with the clathrin mask for HEK 293 cells and treated with 10 μM isoproterenol expressing ( b ) PAmCherry-β1AR, ( c ) PAmCherry-β2AR, ( d ) β-arrestin-2-PAmCherry coexpressed with FLAG-β1AR, ( e ) β-arrestin-2-PAmCherry coexpressed with FLAG-β2AR ( f ) Distribution of diffusion coefficients (D) of false positive detections from HEK 293 cells expressing FLAG-β2AR and imaged under standard sptPALM acquisition conditions to determine contribution of false positive detections in the experimental setup and analysis. ( g ) Distribution of diffusion coefficients (D) of PAmCherry-β2AR, PAmCherry-PLCδ1-PH, and β-arrestin-2-PAmCherry in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13, 21, and 8 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. ( h ) Average MSD plots derived from sptPALM analysis of PAmCherry-β1AR and PAmCherry-β2AR trajectories in HEK 293 cells treated with 10 μM isoproterenol (n=8 and 13 cells, respectively). ( i ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β1AR in live HEK 293 cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13 and 17 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. ( j ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in cells co-expressing FLAG-β1AR and treated with 10 μM isoproterenol (n=13 and 17 cells, respectively). ( k ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β2AR in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=21 and 10 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel d. ( l ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in HEK 293 cells co-expressing FLAG-β2AR and treated with 10 μM isoproterenol (n=21 and 10 cells, respectively). ( m ) Immobile and ( n) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β1AR after 10 μM isoproterenol treatment. ( o ) Immobile and ( p) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β2AR after 10 μM isoproterenol treatment. Trajectory detections are false colored based on the density of detections at each pixel. Error bars represent s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images. ( q ) Proposed cellular pathway for catalytic activation of β-arrestin. ( r ) Representative microscopy images of COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) that were treated with 10 μM isoproterenol for 3 minutes. Then, β-arrestin-2-GFP was photobleached in the indicated yellow region (shown in inset; insets are also shown in ). ( a, b, c, d, e, m, n, o, p, and r ) show representative examples from at least 3 independent experiments. ( f–l ) show data as mean ± s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images; 5 μm for FRAP larger images and 0.5 μm for the insets.

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Generated, Diffusion-based Assay, Expressing, Derivative Assay, Mutagenesis, Marker, Activation Assay, Microscopy

Journal: Nature

Article Title: Catalytic activation of β-arrestin by GPCRs

doi: 10.1038/s41586-018-0079-1

Figure Lengend Snippet: ( a ) Schematic depicting the proposed co-existence of catalytic and scaffolding mechanisms of β-arrestin trafficking tuned according to tail binding affinity, emphasizing the difference in tail versus core interactions (shaded boxes). The tail interaction, requiring GPCR phosphorylation (Rp) drives the scaffold mechanism through its essential role in stable GPCR/β-arrestin complex formation. The core interaction mediates catalysis by providing a kinetically favorable path for β-arrestin to remain captured at the PM irrespective of GPCR dissociation. Such capture requires phosphoinositide binding to the β-arrestin C-domain, explaining why the phosphoinositide requirement is specific to the catalytic mechanism and can be overcome by formation of a sufficiently sufficient stable scaffold complex requiring the phosphorylated GPCR tail. Primary energy inputs maintaining each proposed trafficking cycle are indicated by red arrows. The present results identify a specific requirement of the catalytic mechanism for phosphoinositide binding to the C-domain but they do not exclude binding also in the scaffold complex (which we think is likely). We also cannot presently rule out the possible existence of additional interaction(s) in the catalytic mechanism, such as phosphoinositide binding also to the β-arrestin N-domain that has the potential to displace the β-arrestin C-terminus . ( b ) Representative images (from 3 independent experiments) before and after 10 μM isoproterenol treatment of cells expressing chimeric FLAG-tagged β1AR-V2Rs and imaged live with TIRF microscopy. Profiles of FLAG-β2AR and β-arrestin-2–GFP average enrichment into CCSs in COS-1 cells expressing either an empty vector construct ( c ) or GRK2 ( d ) and treated with 10 μM isoproterenol (n=15 or 12 cells, respectively, from 3 independent experiments). ( e ) Difference in enrichment values between β-arrestin-2–GFP and β2AR from panels c and d showing the effect of GRK2 overexpression. ( f ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β1AR and exposed to 10 μM isoproterenol for the indicated time period. ( g ) Quantification of ERK1/2 activation from the western blots in panel a (n=5 independent experiments, p=0.004 using a one-way ANOVA). ( h ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β2AR and exposed to 10 μM isoproterenol for the indicated time period. ( i ) Quantification of ERK1/2 activation from the western blots in panel c (n=5 independent experiments). ( f ) and ( h ) show representative Western blots from 5 independent experiments. Data shown as mean ± s.e.m. For gel source data, see . Error bars represent s.e.m. ** p < 0.01

Article Snippet: All β-arrestin-2-GFP finger loop proximal mutants were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites HindIII and BbvCI (NEB). β-arrestin-2-GFP (E314K) and β-arrestin-2-GFP (K77E E314K) were created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP or β-arrestin-2-GFP (K77E) construct, respectively. β-arrestin-2-GFP lipid binding mutant was previously described and were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP wild-type plasmid using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP CCS mutant and β-arrestin-2-PAmCherry CCS mutant were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP and β-arrestin-2-PAmCherry plasmids, respectively, using restriction sites BlpI and ApaI (NEB). β-arrestin-2-GFP lipid and CCS mutant and β-arrestin-2-GFP lipid and finger loop proximal mutant constructs were created by inserting a gBlock (IDT) containing the desired mutations into the β-arrestin-2-GFP CCS mutant plasmid and β-arrestin-2-GFP finger loop proximal mutant construct, respectively using restriction sites BbvCI and AhdI (NEB). β-arrestin-2-GFP (L191G, F192G) was created using site-directed mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific) from the wild-type β-arrestin-2-GFP. β-arrestin-1-mVenus finger loop proximal mutant was cloned by inserting a gBlock (IDT) containing the desired mutation into the β-arrestin-1-mVenus wild-type plasmid using restriction sites BamHI and SphI (NEB). β-arrestin-2-GFP KNC mutant was subcloned into β-arrestin-2-GFP from a β-arrestin-2 expression plasmid that was a gift from from Vsevolod Gurevich .

Techniques: Scaffolding, Binding Assay, Expressing, Microscopy, Plasmid Preparation, Construct, Over Expression, Western Blot, Knock-Out, CRISPR, Activation Assay