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J. Biol. Chem., Vol. 281, Issue 18, 12896-12907, May 5, 2006

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Characterization of the Residues in Helix 8 of the Human ₁-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins^*

Noel M. Delos Santos, Lidia A. Gardner, Stephen W. White^¶, and Suleiman W. Bahouth¹

From the Department of Pediatrics, Division of Pediatric Nephrology and Department of Pharmacology, the University of Tennessee Health Sciences Center, Memphis, Tennessee 38163 and ^¶Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

Several key amino acids within amphipathic helix 8 of the human ₁-adrenergic receptor (¹-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly³⁸⁹ or Arg³⁸⁹ allelic variants of the wild type ₁-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type ₁-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp³⁸² and Arg³⁸⁴ within the hydrophilic interface might serve as a tethering site for the G protein.

Received for publication, August 3, 2005 , and in revised form, February 21, 2006.

^* This work was supported by grants from the Le Bonheur Children's Research Center, National Institutes of Health Grant HL 71419, and by a generous gift from Anna and Donald Waite to the Pediatric Nephrology Research Support Fund. 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.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, the 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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