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J. Biol. Chem., Vol. 279, Issue 34, 35326-35333, August 20, 2004

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Subtype-selective Noncompetitive or Competitive Inhibition of Human ₁-Adrenergic Receptors by -TIA^*

Zhongjian Chen, George Rogge, Chris Hague, Dianne Alewood, Barbara Colless, Richard J. Lewis, and Kenneth P. Minneman¶

From the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322 and Xenome Ltd., 50 Meiers Road, Indooroopilly 4068, Queensland, Australia

The 19-amino acid conopeptide (-TIA) was shown previously to antagonize noncompetitively _1B-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human ₁-AR subtypes (_1A, _1B, and _1D). Radioligand binding assays showed that -TIA was 10-fold selective for human _1B-over _1A- and _1D-ARs. As observed with hamster _1B-ARs, -TIA decreased the number of binding sites (B_max) for human _1B-ARs without changing affinity (K_D), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, -TIA had opposite effects at human _1A-ARs and _1D-ARs, decreasing K_D without changing B_max, suggesting it acts competitively at these subtypes. -TIA reduced maximal NE-stimulated [³H]inositol phosphate formation in HEK293 cells expressing human _1B-ARs but competitively inhibited responses in cells expressing _1A- or _1D-ARs. Truncation mutants showed that the amino-terminal domains of _1B- or _1D-ARs are not involved in interaction with -TIA. Alanine-scanning mutagenesis of -TIA showed F18A had an increased selectivity for _1B-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at _1B-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus -TIA noncompetitively inhibits _1B-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at _1B-ARs.

Received for publication, April 2, 2004 , and in revised form, May 17, 2004.

^* This work was supported by grants from the National Institutes of Health (to K. P. M.). 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. Tel.: 404-727-5985; Fax: 404-727-0365; E-mail: kminneman{at}pharm.emory.edu.

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