Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle |
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Institution: | 1. CSIR-National Chemical Laboratory, Pune, India;4. Savitribai Phule Pune University, Pune, India;5. Division of Biology, IISER Pune, Pune, India;1. Research Division, VA Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, United States;2. Department of Physiology, University of Tennessee Health Science Center Memphis, 3N Dunlap Street, Memphis, TN 38163, United States;3. Department of Pharmacology, Graduate School of Pharmaceutical Sciences Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan |
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Abstract: | Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) e.g. β2-adrenoceptor (β2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-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 β2AR and A2BR 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 β2AR- and A2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β2AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A2BR was regulated by GRK5, but not GRK2. β2AR 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 A2BR-AC signalling and attenuated A2BR desensitization, while β2AR-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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