Journal: iScience
Article Title: Robust GRK2/3/6-dependent desensitization of oxytocin receptor in neurons
doi: 10.1016/j.isci.2024.110047
Figure Lengend Snippet: Desensitization of PI(4,5)P 2 hydrolysis and G protein activation, and recruitment of β-arrestin-1/2 and GRK2/3/6 to activated OXTR in primary neuronal cultures (A) Summary graph (bottom) of normalized BRET showing the time course of PI(4,5)P 2 hydrolysis downstream of OXTR activation by 200 nM TGOT (time of application is indicated by an arrow). Schematic (top) shows the BRET configuration with nanoLuc-PH-PLCδ and HaloTag-CAAX. Normalized BRET is reduced rapidly with TGOT application in control (black) followed by quick recovery suggesting desensitization ( n = 3). Gα q inhibitor UBO-QIC (1 μM, blue) abolished the response, whereas Gβγ inhibitor Gallein (40 μM, magenta) had no effect (n = 3–5 per group). (B) Summary graph (bottom) of ΔBRET showing the time course of G protein activation downstream of OXTR activation by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing nanoLuc-Gγ, Gβ, and Gα q along with GRK2-HaloTag. BRET is rapidly increased with TGOT application followed by quick recovery suggesting desensitization at the G protein level (n = 3). (C) Summary graph (bottom) of ΔBRET showing the time course of recruitment of β-arrestin-1 and β-arrestin-2 to neuronal OXTR activated by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing OXTR-nanoLuc and β-arrestin-1 (blue) or β-arrestin-2 (magenta). BRET is increased with TGOT application suggesting recruitment of both β-arrestins to neuronal OXTR (n = 3–5 per group). (D) Summary graph (bottom) of ΔBRET time course shows that GRK2, GRK3, and GRK6 (but not GRK5) are recruited to neuronal OXTR activated by 200 nM TGOT. Schematic (top) shows the BRET configuration with neurons expressing OXTR-nanoLuc and GRK2-HaloTag (red), GRK3-HaloTag (blue), GRK5-HaloTag (gray), or GRK6-HaloTag (magenta) (n = 4 per group). The data in graphs are shown as mean ± SEM.
Article Snippet: The following primary antibodies were used: β-arrestin-1 (Cell Signaling, Cat# 30036), β-arrestin-2 (Cell Signaling, Cat# 3857), GRK2(Santa Cruz Biotechnology, Cat# sc-13143), GRK3(Cell Signaling, Cat# 80362) GRK5(Santa Cruz Biotechnology, Cat# sc-518005), GRK6(Cell Signaling, Cat# 5878) and β-actin (Millipore Sigma, Cat# A1978).
Techniques: Activation Assay, Expressing
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