luciferase-based pglosensor-22f camp reporter plasmid glosensor  (Promega)

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a Map of the PTH1R with sites of three studied Eiken mutations (E35K, Y134S and R485X) shaded red; the site of the Jansen disease mutation (H223R) shaded orange, and sites of C-tail serine phosphorylation shaded blue. b 3-D view of the PTH1R in complex with LA-PTH {cryo-EM structural file PDB.6nBF, ref. } showing the extracellular domain (ECD) and upper portion of the transmembrane domain (TMD) regions of the complex with receptor shaded gray and ligand shaded blue. Sidechain atoms of E35 and Y134 in the PTH1R ECD, and Leu27, Arg19, and Ile5 in the ligand are displayed as spheres with oxygens colored red and nitrogens colored blue. Yellow dashed lines indicate measured distances between side chain oxygen of E35 and the proximal sidechain nitrogen of Arg19 (8.3 Å) and between the side chain oxygen of Y134 and the proximal sidechain carbon of Leu27 (7.9 A˚). c Surface expression levels of the HA-tagged PTH1R variants in transiently transfected Gs22a (HEK293/glosensor) cells measured by immunofluorescence flow cytometry. Mean total cell fluorescence levels are normalized to values in cells transfected with pCDNA3.1. Bar heights indicate means ± SEM of four to six experiments and data points indicate measurements from each separate experiment. P values indicate Student’s t test comparisons to PTH1R-WT. Gating strategies are as described in Supplemental Fig. . d Schematic of a possible mode of PTH1R complexing with PTH(1-34) or PTHrP(1-36) ligand and β-arrestin, which utilizes two key predicted sites of receptor contact involving (1) the N-terminal portion of β-arrestin and phosphorylated (P) serine or threonine residues in the PTH1R C-tail, and (2) the finger loop domain near the center of β-arrestin and the TMD core of the receptor. The inset depicts possible modes of interaction of the receptor with arrestin at the plasma membrane and in endosomes, as suggested by studies on other GPCRs , . The inset graphic was generated using BioRender.com.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Mutagenesis, Cryo-EM Sample Prep, Expressing, Transfection, Immunofluorescence, Flow Cytometry, Fluorescence, Generated

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: a Basal intracellular cAMP generation assessed in Gs22a (HEK293/glosensor) cells transiently transfected with plasmids to express PTH1R-WT, PTH1R-R485X, PTH1R-E35K, PTH1R-Y134S, or PTH1R-H223R, or with pCDNA3.1; shown are changes in glosensor-derived luminescence over time after addition of luciferin ( t = 0). Signals are normalized to the peak signal observed with PTH1R-WT (100%). Peak signals attained for PTH1R-R485X, PTH1R-E35K, PTH1R-Y134S, and PTH1R-H223R, as per-cents of the PTH1R-WT peak, were 719 ± 72 ( P ≤ 0.0001), 163 ± 16 ( P = 0.0023), 111 ± 12 ( P = 0.39) and 756 ± 125 ( P ≤ 0.00036), respectively. b Basal cAMP signaling in Gs22A cells expressing PTH1R - PD (phosphorylation deficient), in which serines at position 489, 491, 492, 493, 494, 496, and 504 in the C-tail are replaced by alanine, and PTHR-E546X, which is truncated at position 546, normalized to PTH1R-WT (100%). c Basal cAMP signaling in Gs22A cells co-transfected with plasmids encoding a PTH1R (WT or mutant) and either pCDNA3.1 or β-arrestin2 YFP ; luminescence signals as counts per second (cps) are plotted vs. time after addition of luciferin (t = 0). The area-under-the-curve (AUC) of the responses for co-transfection with pCDNA3.1 vs. with β-arrestin2 YFP , normalized to the response for PTH1R-WT/pCDNA3.1, for PTH1R-1WT were 100 ± 0 vs. 84 ± 23, P = 0.5; for PTH1R-R485X were 602 ± 36 vs.150 ± 19, P = 0.0004, and for PTH1R-H223R were 466 ± 208 vs. 381 ± 170, P = 0.8 ( P values determined by Student’s t test). Data are means (±SEM) of six ( a ) or three independent experiments with three or more wells in each.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Derivative Assay, Expressing, Mutagenesis, Cotransfection

Journal: Communications Biology

Article Title: Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome

doi: 10.1038/s42003-023-04966-0

Figure Lengend Snippet: cAMP Signaling responses to PTH and PTHrP ligands were assessed in Gs22a (HEK293/glosensor) cells transiently transfected with PTH1R-WT or a PTH1R mutant. a Dose-responses for PTH(1-34) and PTHrP(1-36). For each ligand concentration on each receptor, the peak luminescence signal, which typically occurred ~15 min after ligand addition, was normalized to the maximum peak signal observed for that ligand on PTH1R-WT (100%) and plotted vs. ligand concentration. b Time courses of cAMP signaling following addition of PTH(1-34) or PTHrP(1-36) at concentrations of 3 nM. The luminescence signals are normalized to the peak signal observed for each ligand on PTH1R-WT (100%); the signals observed for PTHrP(1-36) on PTH1R-E35K at 12–16 min were higher than those on PTH1R-WT (* p < 0.05). c Time courses of cAMP signaling following washout to remove unbound ligand of the cells treated in b . The cAMP-dependent luminescence signals observed for each ligand after washout are normalized to the peak signal observed after washout on PTH1R-WT (100%). d Cells were pretreated for 30 min with Ile 5 -PTHrP(1-36) or PTHrP(1-36), each at a concentration of 10 nM, and then after washout, cAMP-dependent luminescence signals were recorded and normalized to the peak signal observed for each ligand after washout on PTH1R-WT (100%). Data are means (±SEM) of five ( a ), four ( b , c , e ) or three ( d ) separate experiments.

Article Snippet: Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.) .

Techniques: Transfection, Mutagenesis, Concentration Assay

Journal: JBMR Plus

Article Title: Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism

doi: 10.1002/jbm4.10604

Figure Lengend Snippet: Recruitment of β‐arrestin and signaling via the Gq/PLC/IP 3 /iCa 2+ pathway. ( A ) GBR‐24 (HEK293/GloSensor/β ‐ arrestin2 YFP stable) cells ( ³⁰ ) were transiently transfected to express the WT or a mutant PTH1R and then treated on coverslips with PTH(1‐34) TMR (30nM) for 30 minutes at room temperature. The cells were then rinsed, fixed, stained with DAPI, and imaged using a fluorescence microscope (magnification = ×400). Areas enclosed in dashed boxes are shown digitally enlarged 7×. ImageJ‐derived colocalization plots of β‐arrestin2 YFP and with PTH(1‐34) TMR in individual cells, with corresponding Pearson correlation coefficients ( R ), are shown in the rightmost columns. ( B ) Analysis of iCa 2+ signaling was assessed in transiently transfected GS‐22a cells using the Ca‐sensitive fluorophore Fura2‐AM. After preloading the cells with Fura2‐AM, baseline ratiometric fluorescence (sequential excitation at 340 nm and 380; emission at 515 nm) was measured in a PerkinElmer Envision plate reader for 20 seconds prior to (baseline) and for 140 seconds after addition of PTH(1‐34) or PTHrP(1‐36) each at a concentration of 100nM. Shown are the means (±SEM) of data from five or four (PTHrP(1‐36) ligand) separate experiments.

Article Snippet: GS‐22a cells are derived from HEK293 cells (American Type Culture Collection [ATCC], Manassas, VA, USA; CRL‐1573) by stably transfection with the luciferase‐based GloSensor (Promega, San Luis Obispo, CA, USA) cAMP reporter plasmid pGloSensor‐22F.

Techniques: Transfection, Mutagenesis, Staining, Fluorescence, Microscopy, Derivative Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: The COMMD3/8 complex determines GRK6 specificity for chemoattractant receptors

doi: 10.1084/jem.20181494

Figure Lengend Snippet: COMMD8 promotes β-arrestin–mediated signaling of CXCR4. (A) IB analysis for Gα i activation induced by CXCL12 in control and Commd8 Δ B cells. (B) CXCR4-mediated inhibition of cAMP production was assessed by the GloSensor reporter in vector-transfected control and COMMD8-deficient (sg COMMD8 ) HEK293 cells expressing Flag-tagged CXCR4. The amounts of cAMP are plotted as fold increases of luminescence over the levels in unstimulated cells. Data are shown as the mean ± SD of triplicates and representative of three independent experiments. (C) Intracellular calcium responses to CXCL12 in control and Commd8 Δ B cells. Responses are plotted as the ratio of Cal-520 to Fura Red fluorescence. Data are representative of two independent experiments. (D) IP assay for the recruitment of endogenous β-arrestin-2 to Flag-tagged CXCR4 in vector-transfected control and COMMD8-deficient (sg Commd8 ) 2PK-3 cells after stimulation with CXCL12. (E–H) IB analysis for the phosphorylation of ERK (E and G) and p38 (F and H) in Commd8 Δ (E and F) and Arrb2 −/− (G and H) B cells after stimulation with CXCL12. B cells from littermate Commd8 f/+ Mb1 Cre/+ and Commd8 f/f Mb1 +/+ (E and F) or Arrb2 +/+ (G and H) mice served as the control. Error bars represent the mean ± SD of three (A and E–H) or four (D) independent experiments, and representative blots are shown. *, P < 0.05; **, P < 0.01; ns, not significant. The P values were obtained by two-tailed unpaired (A and D–H) or paired (B) t test. Asterisk indicates nonspecific bands (A, D, and H). coIP, coimmunoprecipitation.

Article Snippet: To assess CXCR4-mediated inhibition of cAMP production, control and COMMD8- or COMMD3-deficient HEK293 cells were transfected with plasmids encoding Flag-tagged CXCR4 and a luciferase-based cAMP biosensor, GloSensor (pGloSensor-22F; Promega) at a 1:1 ratio.

Techniques: Activation Assay, Inhibition, Plasmid Preparation, Transfection, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: The COMMD3/8 complex determines GRK6 specificity for chemoattractant receptors

doi: 10.1084/jem.20181494

Figure Lengend Snippet: COMMD8 is involved in β-arrestin-mediated signaling of β 2 AR. (A) Confocal microscopy for the subcellular localization and colocalization of Flagg-tagged β 2 AR (red) and Myc-tagged COMMD8 (green) in HEK293 cells before and at 1 min after treatment with pan-βAR agonist isoproterenol. Colocalization of the signals (arrowheads) was quantified as in . Each symbol represents an individual cell, and bars indicate means (0 min, n = 20; 1 min, n = 20). Representative images are shown. Bar, 10 µm. (B) IP assay for the interaction of Myc-tagged COMMD8 with Flag-tagged β 2 AR in 2PK-3 cells after stimulation with selective β 2 AR agonist clenbuterol. (C) cAMP production assessed by GloSensor in vector-transfected control and COMMD8-deficient (sg COMMD8 ) HEK293 cells after stimulation with isoproterenol. The amounts of cAMP are plotted as fold increases of luminescence over the levels in unstimulated cells. Data are shown as the mean ± SD of triplicates and representative of three experiments. (D) IP assay for the recruitment of endogenous β-arrestin-2 to Flag-tagged β 2 AR in vector-transfected control and COMMD8-deficient (sg Commd8 ) 2PK-3 cells after stimulation with isoproterenol. Asterisks indicate nonspecific bands. (E) IB analysis for the phosphorylation of ERK in vector-transfected control and sg Commd8 2PK-3 cells after stimulation with isoproterenol. (F) IB analysis for GRK6-mediated phosphorylation (p) at S355/356 of Flag-tagged β 2 AR in vector-transfected control and sg Commd8 2PK-3 cells after stimulation with isoproterenol. Error bars represent the mean ± SD of three (B), four (E and F), or five (D) independent experiments, and representative blots are shown. (G) Tango assay for the association of COMMD8 with β 2 AR in the presence of paroxetine. (H) Tango assay for the association of COMMD8 with a C terminus–truncated β 2 AR mutant lacking GRK2 phosphorylation sites. The luminescence from the tTA-dependent firefly luciferase reporter was normalized to that from the cotransfected Renilla luciferase and plotted as fold increases over the levels in unstimulated cells (G and H). *, P < 0.05; **, P < 0.01; ns, not significant. The P values were obtained by two-tailed unpaired (A and D–H) or paired (B and C) t test. coIP, coimmunoprecipitation; DIC, differential interference contrast.

Article Snippet: To assess CXCR4-mediated inhibition of cAMP production, control and COMMD8- or COMMD3-deficient HEK293 cells were transfected with plasmids encoding Flag-tagged CXCR4 and a luciferase-based cAMP biosensor, GloSensor (pGloSensor-22F; Promega) at a 1:1 ratio.

Techniques: Confocal Microscopy, Plasmid Preparation, Transfection, Mutagenesis, Luciferase, Two Tailed Test