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Fig. 1. GRK2 and <t>GRK3,</t> but not GRK5 or GRK6, rapidly desensitize group I mGluR-mediated Ca2+ responses. (A) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation. (B) Summary bar graph showing effects of GRK coexpression on FWHM. (C) Zoom-in to represent ative traces showing the effect of GRK2 on ON kinetics of R-GECO response. (D) Summary bar graph showing the effects of GRK coexpression on rise time. (E and F) Same as (A) and (B), but for mGluR5. (G) Representative individual cell traces showing oscillatory responses, which are reduced with GRK2 coexpression. (H) Summary bar graph showing the effects of GRKs on the proportion of cells showing oscillatory responses to glutamate. (I) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation in parental versus GRK2/3 double KO cells. (J) Summary bar graph showing the effects of GRK2/3 double KO and GRK2 rescue on FWHM. (K) Summary histogram showing the distribution of FWHM values across individual cells for each condition and replicate in (J). Fifty to 80 cells per experiment are used to produce a normalized histogram that is averaged across separate experiments to produce mean histograms with error bars. GRK2/3 KO pro- duces a shift in the distribution toward higher FWHM values, while GRK2 rescue produces dose-dependent shifts toward lower values and a narrower distribution. In bar graphs, each point represents an independent measurement; all graphs come from two to five separate experimental days, and error bars represent SEM. One-way ANOVA with Tukey’s multiple comparisons tests. *P < 0.05, **P < 0.01, and ***P < 0.001.
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Fig. 1. GRK2 and <t>GRK3,</t> but not GRK5 or GRK6, rapidly desensitize group I mGluR-mediated Ca2+ responses. (A) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation. (B) Summary bar graph showing effects of GRK coexpression on FWHM. (C) Zoom-in to represent ative traces showing the effect of GRK2 on ON kinetics of R-GECO response. (D) Summary bar graph showing the effects of GRK coexpression on rise time. (E and F) Same as (A) and (B), but for mGluR5. (G) Representative individual cell traces showing oscillatory responses, which are reduced with GRK2 coexpression. (H) Summary bar graph showing the effects of GRKs on the proportion of cells showing oscillatory responses to glutamate. (I) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation in parental versus GRK2/3 double KO cells. (J) Summary bar graph showing the effects of GRK2/3 double KO and GRK2 rescue on FWHM. (K) Summary histogram showing the distribution of FWHM values across individual cells for each condition and replicate in (J). Fifty to 80 cells per experiment are used to produce a normalized histogram that is averaged across separate experiments to produce mean histograms with error bars. GRK2/3 KO pro- duces a shift in the distribution toward higher FWHM values, while GRK2 rescue produces dose-dependent shifts toward lower values and a narrower distribution. In bar graphs, each point represents an independent measurement; all graphs come from two to five separate experimental days, and error bars represent SEM. One-way ANOVA with Tukey’s multiple comparisons tests. *P < 0.05, **P < 0.01, and ***P < 0.001.
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Fig. 1. GRK2 and <t>GRK3,</t> but not GRK5 or GRK6, rapidly desensitize group I mGluR-mediated Ca2+ responses. (A) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation. (B) Summary bar graph showing effects of GRK coexpression on FWHM. (C) Zoom-in to represent ative traces showing the effect of GRK2 on ON kinetics of R-GECO response. (D) Summary bar graph showing the effects of GRK coexpression on rise time. (E and F) Same as (A) and (B), but for mGluR5. (G) Representative individual cell traces showing oscillatory responses, which are reduced with GRK2 coexpression. (H) Summary bar graph showing the effects of GRKs on the proportion of cells showing oscillatory responses to glutamate. (I) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation in parental versus GRK2/3 double KO cells. (J) Summary bar graph showing the effects of GRK2/3 double KO and GRK2 rescue on FWHM. (K) Summary histogram showing the distribution of FWHM values across individual cells for each condition and replicate in (J). Fifty to 80 cells per experiment are used to produce a normalized histogram that is averaged across separate experiments to produce mean histograms with error bars. GRK2/3 KO pro- duces a shift in the distribution toward higher FWHM values, while GRK2 rescue produces dose-dependent shifts toward lower values and a narrower distribution. In bar graphs, each point represents an independent measurement; all graphs come from two to five separate experimental days, and error bars represent SEM. One-way ANOVA with Tukey’s multiple comparisons tests. *P < 0.05, **P < 0.01, and ***P < 0.001.
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Fig. 1. GRK2 and GRK3, but not GRK5 or GRK6, rapidly desensitize group I mGluR-mediated Ca2+ responses. (A) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation. (B) Summary bar graph showing effects of GRK coexpression on FWHM. (C) Zoom-in to represent ative traces showing the effect of GRK2 on ON kinetics of R-GECO response. (D) Summary bar graph showing the effects of GRK coexpression on rise time. (E and F) Same as (A) and (B), but for mGluR5. (G) Representative individual cell traces showing oscillatory responses, which are reduced with GRK2 coexpression. (H) Summary bar graph showing the effects of GRKs on the proportion of cells showing oscillatory responses to glutamate. (I) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation in parental versus GRK2/3 double KO cells. (J) Summary bar graph showing the effects of GRK2/3 double KO and GRK2 rescue on FWHM. (K) Summary histogram showing the distribution of FWHM values across individual cells for each condition and replicate in (J). Fifty to 80 cells per experiment are used to produce a normalized histogram that is averaged across separate experiments to produce mean histograms with error bars. GRK2/3 KO pro- duces a shift in the distribution toward higher FWHM values, while GRK2 rescue produces dose-dependent shifts toward lower values and a narrower distribution. In bar graphs, each point represents an independent measurement; all graphs come from two to five separate experimental days, and error bars represent SEM. One-way ANOVA with Tukey’s multiple comparisons tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

Journal: Science advances

Article Title: Control of Gα q signaling dynamics and GPCR cross-talk by GRKs.

doi: 10.1126/sciadv.abq3363

Figure Lengend Snippet: Fig. 1. GRK2 and GRK3, but not GRK5 or GRK6, rapidly desensitize group I mGluR-mediated Ca2+ responses. (A) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation. (B) Summary bar graph showing effects of GRK coexpression on FWHM. (C) Zoom-in to represent ative traces showing the effect of GRK2 on ON kinetics of R-GECO response. (D) Summary bar graph showing the effects of GRK coexpression on rise time. (E and F) Same as (A) and (B), but for mGluR5. (G) Representative individual cell traces showing oscillatory responses, which are reduced with GRK2 coexpression. (H) Summary bar graph showing the effects of GRKs on the proportion of cells showing oscillatory responses to glutamate. (I) Representative normalized average traces showing kinetics of glutamate-induced R-GECO response to mGluR1 activation in parental versus GRK2/3 double KO cells. (J) Summary bar graph showing the effects of GRK2/3 double KO and GRK2 rescue on FWHM. (K) Summary histogram showing the distribution of FWHM values across individual cells for each condition and replicate in (J). Fifty to 80 cells per experiment are used to produce a normalized histogram that is averaged across separate experiments to produce mean histograms with error bars. GRK2/3 KO pro- duces a shift in the distribution toward higher FWHM values, while GRK2 rescue produces dose-dependent shifts toward lower values and a narrower distribution. In bar graphs, each point represents an independent measurement; all graphs come from two to five separate experimental days, and error bars represent SEM. One-way ANOVA with Tukey’s multiple comparisons tests. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following cDNA for bovine GRKs were used: untagged GRK2 (Addgene #14691), GRK3 (Addgene #32689), GRK5 (Addgene #14690), GRK6 (Addgene #32693), GRK2-GFP (24), and GRK2-CAAX (Addgene #166224).

Techniques: Activation Assay