QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.106.025346v1 70/3/838    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zeitoun, O. Articles by Bahouth, S. W. Search for Related Content PubMed PubMed Citation Articles by Zeitoun, O. Articles by Bahouth, S. W.

Mutagenesis within Helix 6 of the Human ₁-Adrenergic Receptor Identifies Lysine³²⁴ as a Residue Involved in Imparting the High-Affinity Binding State of Agonists

Department of Pharmacology, the University of Tennessee Health Sciences Center, Memphis, Tennessee (O.Z., N.M.D., L.A.G., S.W.B.); and Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee (S.W.W.)

Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human ₁-adrenergic receptor (₁-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT ₁-AR, intrinsic activities of full and partial agonists in activating the Lys³²⁴ Ala ₁-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT ₁-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT ₁-AR or the K324A mutant to G_s, there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of G_s, which acts a GDP-bound G_s mimic, to high- and low-affinity sites. Based upon the three-dimensional architecture of the human ₁-AR, the distance between Lys³²⁴ and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys³²⁴ lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDP-bound isoform of G_s.

Address correspondence to: Suleiman W. Bahouth, Department of Pharmacology, The University of Tennessee Health Sciences center, 874 Union Avenue, Memphis, TN 38163. E-mail: sbahouth{at}utmem.edu

This article has been cited by other articles:

				S. Gavi, D. Yin, E. Shumay, H.-y. Wang, and C. C. Malbon Insulin-Like Growth Factor-I Provokes Functional Antagonism and Internalization of {beta}1-Adrenergic Receptors Endocrinology, June 1, 2007; 148(6): 2653 - 2662. [Abstract] [Full Text] [PDF]

				L. A. Gardner, A. P. Naren, and S. W. Bahouth Assembly of an SAP97-AKAP79-cAMP-dependent Protein Kinase Scaffold at the Type 1 PSD-95/DLG/ZO1 Motif of the Human beta1-Adrenergic Receptor Generates a Receptosome Involved in Receptor Recycling and Networking J. Biol. Chem., February 16, 2007; 282(7): 5085 - 5099. [Abstract] [Full Text] [PDF]