Differential regulation of β 2 -adrenoceptor and adenosine A 2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle.

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.
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