RH domain of GRK2 in GPCRs desensitization

MAIA CABRERA; NATALIA FERNANDEZ; EMILIANA ECHEVERRÍA; SONIA RIPOLL; BURGHI, VALERIA; FEDERICO MONKZOR

Bariloche

Simposio; Fourth South American Symposium in Signal and Molecular Medicine; 2018

G protein coupled receptor kinase 2 (GRK2) plays a major role in GPCRs signalling regulation by phosphorylation of receptor and uncoupling from G protein. Beyond this canonical model, several evidences support a kinase independent desensitization process, mediated by GRK2 RGS homology domain (RH). The aim of this work was to deepen the underlying mechanisms of RH-mediated regulation of GPCR signalling.RH-dependent desensitization was observed for both, Gαq and Gαs coupled GPCRs by functional assays performed in naïve or transfected systems. Our results showed that calcium response of histamine receptor type 1 (H1R), and cAMP response of histamine receptor type 2 (H2R) or β3 adrenergic receptor (B3AR) are significantly increased in presence of an RH inactive GRK2 dominant negative mutant.Consistently, we observed a RH-GFP recruitment to areas of active signalling on plasma membrane of HELA cells when cotransfected with constitutive active Gαq. Such translocation was observed also when H2R is overexpressed, suggesting a potential interaction between GRK2 and Gαs subunit. Accordingly, we performed a co-immunoprecipitation assay in HEK293T cells cotransfected with HA-tagged Gαs, kinase inactive GRK2 and H2R. Our results indicate a specific GRK2-Gαs interaction under basal conditions and stimulation of the receptor in a conserved cellular context. Moreover, the interaction is also observed when HEK293T endogenous BARs are stimulated with isoproterenol.In conclusion, we advanced in the understanding of GPCRs desensitization mechanism mediated by GRK2, suggesting that RH domain is able to impede GPCRs response through a direct modulation at G protein level that is independent of receptor phosphorylation.