Abstract
There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.
Highlights
The 2-adrenergic receptor (2AR)2 is a member of the extended G protein-coupled receptor (GPCR) family
G proteincoupled receptor kinase family (GRKs)-mediated phosphorylation of residues within the receptor cytoplasmic tail triggers the translocation of nonvisual arrestins (-arrestin 1 and -arrestin 2) to the cell surface where they interact with phosphorylated receptors, causing receptor uncoupling from stimulatory G protein [2,3,4]. -Arrestins interact directly with two structural components of clathrin-coated pits, clathrin and AP-2, which promote the endocytosis of 2ARs into early endosomes via clathrin-coated vesicles [5,6,7]
We used receptors in which only the putative GRK sites (Ser-355, -356, and -364) have been mutated to either alanine (2AR-SA3) to eliminate phosphorylation or to aspartic acid (2AR-SD3), a substitution that may mimic the phosphorylated state. To accomplish these goals we studied the effects of GRK site mutations on receptor desensitization, internalization, cellular trafficking, and EGFP--arrestin 2 translocation
Summary
The 2-adrenergic receptor (2AR) is a member of the extended G protein-coupled receptor (GPCR) family. Receptor internalization is significantly impaired, suggesting that phosphorylation of these sites is essential for high affinity -arrestin interaction In these studies we utilized 2ARs in which the PKA consensus sites were mutated and a 6-histidine tag was added to the carboxyl terminus [9], modifications that may independently alter receptor desensitization or cellular localization [12]. We used receptors in which only the putative GRK sites (Ser-355, -356, and -364) have been mutated to either alanine (2AR-SA3) to eliminate phosphorylation or to aspartic acid (2AR-SD3), a substitution that may mimic the phosphorylated state To accomplish these goals we studied the effects of GRK site mutations on receptor desensitization, internalization, cellular trafficking, and EGFP--arrestin 2 translocation. Our data provide insights into the 2AR domains governing -arrestin interaction, receptor internalization, and desensitization
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