Abstract
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. 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) , KGGFSFRF-NH2 , was docked in this GPR103 model and the ligand-receptor complex was submitted to energy minimization. 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. 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.
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