G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although b3-adrenergic receptor (b3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce b3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of b3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human b3AR presented a short-term desensitization of cAMP response stimulated by the b3AR agonist, BRL37344, and not by forskolin. We found that b3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased b3AR desensitization. Consistently, stimulation of b3AR increased interaction between GRK2 and Gas subunit. Furthermore, in rat cardiomyocytes endogenously expressing b3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the b3AR in heart function and disease.
G protein‐coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2‐mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates’ ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation‐independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.
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