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 β3-adrenergic receptor (β3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce β3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of β3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human β3AR presented a short-term desensitization of cAMP response stimulated by the β3AR agonist, BRL37344, and not by forskolin. We found that β3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased β3AR desensitization. Consistently, stimulation of β3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing β3AR, 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 β3AR in heart function and disease.
Highlights
B3-adrenergic receptor (b3AR) was the last member of the b adrenoreceptors to be identified (Emorine et al, 1989) and presents a more restrictive expression profile than their counterparts
B3AR has been thought to be insensitive to protein kinase (PKA) or G protein coupled receptor kinase (GRK) mediated desensitization due to the lack of potential phosphorylation sites in its third intracellular loop and c-terminal tail that are present in b1AR and b2AR variants
Results presented in this work show that human b3-adrenergic receptor (b3AR) heterologously expressed in HEK293T cells is sensitive to short-term desensitization by a mechanism that takes place upstream adenylyl cyclase (AC) activation and does not involve Gas downregulation nor a switch in receptor coupling to Gai
Summary
B3-adrenergic receptor (b3AR) was the last member of the b adrenoreceptors to be identified (Emorine et al, 1989) and presents a more restrictive expression profile than their counterparts. Studies carried out in diverse experimental models have shown that agonist-induced b3AR desensitization occurs (Chaudhry and Granneman, 1994; Scarpace et al, 1999; Hutchinson et al, 2000; Vrydag et al, 2009), and that long term treatment with isoproterenol induced b3AR-mediated cAMP response desensitization in HEK293 transfected cells by reducing the activity of adenylyl cyclase (Vrydag et al, 2009; Michel-Reher and Michel, 2013; Okeke et al, 2019) In this context, we aimed to evaluate agonist induced short-term desensitization of human b3AR expressed in HEK293 cells, and the underlying mechanism. In rat cardiomyocytes that natively express b3AR, dominant negative mutant of the RH domain of GRK2 increased cAMP response to BRL37344 and attenuated desensitization
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