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Molecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure-activity relationship studies.

ABSTRACT: BACKGROUND AND PURPOSE: The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand-receptor interactions by site-directed mutagenesis of GPR103 and designed point-substituted 26RFa analogues. EXPERIMENTAL APPROACH: Using the X-ray structure of the ?2 -adrenoceptor, a 3-D molecular model of GPR103 has been built. The bioactive C-terminal octapeptide 26RFa(19-26) , KGGFSFRF-NH2 , was docked in this GPR103 model and the ligand-receptor complex was submitted to energy minimization. KEY RESULTS: In the most stable complex, the Phe-Arg-Phe-NH2 part was oriented inside the receptor cavity, whereas the N-terminal Lys residue remained outside. A strong intermolecular interaction was predicted between the Arg(25) residue of 26RFa and the Gln(125) residue located in the third transmembrane helix of GPR103. To confirm this interaction experimentally, we tested the ability of 26RFa and Arg-modified 26RFa analogues to activate the wild-type and the Q125A mutant receptors transiently expressed in CHO cells. 26RFa (10(-6) ?M) enhanced [Ca(2+) ]i in wild-type GPR103-transfected cells, but failed to increase [Ca(2+) ]i in Q125A mutant receptor-expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA(25) ]26RFa(20-26) , that was unable to activate the wild-type GPR103, but antagonized 26RFa-evoked [Ca(2+) ]i increase. CONCLUSION AND IMPLICATIONS: Altogether, these data provide strong evidence for a functional interaction between the Arg(25) residue of 26RFa and the Gln(125) residue of GPR103 upon ligand-receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.

SUBMITTER: Neveu C

PROVIDER: S-EPMC4209149 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Molecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure-activity relationship studies.

Neveu C C Dulin F F Lefranc B B Galas L L Calbrix C C Bureau R R Rault S S Chuquet J J Boutin J A JA Guilhaudis L L Ségalas-Milazzo I I Vaudry D D Vaudry H H Santos J Sopkova-de Oliveira JS Leprince J J

British journal of pharmacology 20140905 19

<h4>Background and purpose</h4>The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand-receptor interactions by site-directed mutagenesis of GPR103 and designed point-substituted 26RFa analogues.<h4>Experimental approach</h4>Using the X-ray structure of the β2 -adrenoceptor, a 3-D molecular model of GPR103 has been built. The bioactive C-terminal octapeptide 26RFa(19-26) ...[more]

PMID: 24913445

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Project description:The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.

Dataset Information

Molecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure-activity relationship studies.

Publications

Molecular basis of agonist docking in a human GPR103 homology model by site-directed mutagenesis and structure-activity relationship studies.

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