The Role of Sequestration in G Protein-coupled Receptor Resensitization

doi:10.1074/jbc.272.1.5

The Role of Sequestration in G Protein-coupled Receptor Resensitization

REGULATION OF β₂-ADRENERGIC RECEPTOR DEPHOSPHORYLATION BY VESICULAR ACIDIFICATION*

From the Departments of Medicine and Biochemistry, Howard Hughes Medical Institute at Duke University Medical Center, Durham, North Carolina 27710

^§ To whom correspondence should be addressed. Tel.: 919-684-2974; Fax: 919-684-8875.

Abstract

G protein-coupled receptor kinases phosphorylate the agonist occupied conformation of G protein-coupled receptors in the plasma membrane, leading to their desensitization. Receptor resensitization requires receptor dephosphorylation, a process which is mediated by a plasma and vesicular membrane-associated form of PP-2A. We present evidence that, like receptor phosphorylation, receptor dephosphorylation is tightly regulated, requiring a specific receptor conformation induced by vesicular acidification. In vitro, spontaneous dephosphorylation of phosphorylated receptors is observed only at acidic pH. Furthermore, in intact cells upon agonist stimulation, phosphorylated receptors traffic from the plasma membrane to vesicles where they become physically associated with the phosphatase and dephosphorylated. Treatment of cells with NH₄Cl, which disrupts the acidic pH found in endosomal vesicles, blocks association of the receptors with the phosphatase and blocks receptor dephosphorylation. These findings suggest that a conformational change in the receptor induced by acidification of the endosomal vesicles is the key determinant regulating receptor dephosphorylation and resensitization.

Footnotes

↵^‡ These authors contributed equally to this work.
↵* This work was supported by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

β₂AR

β₂-adrenergic receptor(s)

GRK

G protein-coupled receptor kinase

GRP

G protein-coupled receptor phosphatase

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

PAGE

polyacrylamide gel electrophoresis.
- Received October 8, 1996.
- Revision received November 4, 1996.