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Originally published In Press as doi:10.1074/jbc.M201815200 on April 26, 2002

J. Biol. Chem., Vol. 277, Issue 28, 25519-25526, July 12, 2002
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Vasoactive Intestinal Polypeptide Type-1 Receptor Regulation
DESENSITIZATION, PHOSPHORYLATION, AND SEQUESTRATION*

Michael A. ShetzlineDagger , Julia K. L. Walker§, Kenneth J. Valenzano, and Richard T. Premont

From the Departments of Cell Biology and Medicine, Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina 27710

The vasoactive intestinal polypeptide type-1 (VPAC1) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC1 receptor are largely unknown. We examined agonist-dependent VPAC1 receptor signaling, phosphorylation, desensitization, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5-10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC1 receptor (VPAC1R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC1 receptor revealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stimulated internalization of the VPAC1R peaked in 10 min, and neither overexpressed beta -arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC1 receptor internalization. Interestingly, VPAC1R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC1R that did demonstrate dose-dependent GRK specificity. Therefore, VPAC1 receptor regulation involves agonist-stimulated, GRK-mediated phosphorylation, beta -arrestin translocation, and dynamin-dependent receptor internalization. Moreover, study of endogenously expressed receptors may provide information not evident in overexpressed systems.

* This work was supported in part by National Institutes of Health Grants 5T32DK07568 and DK02544 and by the Mal Tyor Scholarship Award in Gastroenterology (to M. A. S.).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.

Dagger To whom correspondence should be addressed: Duke University Medical Center, Box 3083, Durham, NC 27710. Tel.: 919-681-6814; Fax: 919-684-4983; E-mail: shetz001@mc.duke.edu.

§ Recipient of an Medical Research Council/Canadian Lung Association postdoctoral fellowship.

Present address: Purdue Pharma, L.P., Cranbury, NJ 08512.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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