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J. Biol. Chem., Vol. 279, Issue 20, 21135-21143, May 14, 2004

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Role of the Cyclic AMP-dependent Protein Kinase in Homologous Resensitization of the ₁-Adrenergic Receptor^*

Lidia A. Gardner, Noel M. Delos Santos, Shannon G. Matta, Michael A. Whitt, and Suleiman W. Bahouth¶

From the Pharmacology and Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163

A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human ₁-adrenergic receptor (₁-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) ₁-AR recycled with a t_0.5 of 14 ± 3 min. Mutagenesis of Ser³¹² to alanine (Ser³¹² Ala ₁-AR) or to the phosphoserine mimic aspartic acid (Ser³¹² Asp ₁-AR) resulted in ₁-AR constructs that were pharmacologically indistinguishable from the WT ₁-AR. The internalized Ser³¹² Asp ₁-AR recycled efficiently with a t_0.5 of 11 ± 3 min, whereas the internalized Ser³¹² Ala ₁-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized ₁-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT ₁-AR. Phosphorylation studies revealed that the ₁-AR is partially phosphorylated by PKA and that phosphorylation of the ₁-AR by the catalytic subunit of PKA occurs exclusively at Ser³¹². Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.

Received for publication, December 12, 2003 , and in revised form, February 17, 2004.

^* This work was supported by a grant-in-aid from the Southeastern affiliate of the American Heart Association (to S. W. B.) and National Institutes of Health National Service Research Award Grant F32-HL 71419 (to N. M. D.). The confocal microscopy facility is funded by National Institutes of Health grant S10 RR13725 (to M. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Tennessee Health Sciences Center, 874 Union Ave., Memphis, TN 38163. Tel.: 901-448-1503; Fax: 901-448-7206; E-mail: sbahouth{at}utmem.edu.

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