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The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.

ABSTRACT: BACKGROUND AND PURPOSE: Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding. EXPERIMENTAL APPROACH: Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human alpha(2A)-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays. KEY RESULTS: Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse alpha(2A)-adrenoceptors were observed for mutations at 5.43, xl2.49 and xl2.51. The binding profile of RS-79948-197 was reversed only by the XL2 substitutions. CONCLUSIONS AND IMPLICATIONS: Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse alpha(2A)-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human alpha(2A)-adrenoceptor. Thus, XL2 is involved in determining the species preferences of alpha(2A)-adrenoceptors of human and mouse for some antagonists.

SUBMITTER: Laurila JM

PROVIDER: S-EPMC2189838 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.

Laurila J M M JM Xhaard H H Ruuskanen J O JO Rantanen M J M MJ Karlsson H K HK Johnson M S MS Scheinin M M

British journal of pharmacology 20070611 8

<h4>Background and purpose</h4>Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and ma ...[more]

PMID: 17558432

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Project description:The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.

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The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.

Publications

The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.

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