The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor

Abstract

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.

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling

“…In this sense, GRK2 interaction with Gα has been demonstrated by other groups 5,30 and by us (Ref. [6] and Figure 5A).…”

“…GRK2 is a protein with a confirmed therapeutic potential for several pathologies such as hypertension, heart failure, Alzheimer's, multiple sclerosis, and rheumatoid arthritis. Although its kinase domain has been extensively studied in the search for inhibitors, during the past years its RGS homology domain has also been postulated as an interesting target to be modulated 6,27,32,33,34 . RGS family members have been under study, and several protein inhibitors have been found.…”

“…cAMP content was determined by the competition of [ 3 H]-cAMP for PKA in a cAMP radiobinding protein assay, as previously described. 6 Two replicates per condition were performed in each independent experiment.…”

“…It has been described that the RH domain of GRK2 is able to interact with G proteins to avoid downstream signaling in a mechanism independent of receptor phosphorylation. 5,6,30 In this context, we evaluated the effect of the inhibitors on the direct interaction between GRK2 and the Gαs protein, performing co-immunoprecipitation of both proteins in whole-cell assays.…”

“…The RH domain is present at the N-terminal of GRK2 and is capable of binding directly to the G protein, causing its inactivation and preventing subsequent signaling. [3][4][5][6] Desensitization of GPCRs signaling in a kinase-independent manner has been described for several GPCRs with varied couplings: adenosine type 1 receptor and μ-opioid receptor, 7 prostaglandin E2 receptor, 8 histamine H1 and H2 receptors (H1R and H2R), 9,10 lysophosphatidic acid (LPA1) receptor, 11 endothelin receptor type A and B, 12,13 follicle-stimulating hormone receptor, 14 and D2 dopamine receptor 15 among many others. In some cases, expression of the RH domain of GRK2 resulted sufficient for attenuating receptor signaling.…”