Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Abstract Generation of cAMP through G s -coupled G protein-coupled receptor (GPCR) [ e.g. β 2 -adrenoceptor (β 2 AR), adenosine A 2B receptor (A 2B R)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G s -coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β 2 AR and A 2B R can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β 2 AR- and A 2B R-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β 2 AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A 2B R was regulated by GRK5, but not GRK2. β 2 AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A 2B R-AC signalling and attenuated A 2B R desensitization, while β 2 AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.