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 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. 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 Arg25 residue of 26RFa and the Gln125 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 [Ca2+]i in wild‐type GPR103‐transfected cells, but failed to increase [Ca2+]i in Q125A mutant receptor‐expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA25]26RFa(20–26), that was unable to activate the wild‐type GPR103, but antagonized 26RFa‐evoked [Ca2+]i increase. Conclusion and Implications Altogether, these data provide strong evidence for a functional interaction between the Arg25 residue of 26RFa and the Gln125 residue of GPR103 upon ligand‐receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.

The Arg–Phe‐amide peptide 26RFa/glutamine RF‐amide peptide and its receptor: IUPHAR Review 24

“…Structure of the final model of the human QRFP receptor monomer after energy minimization (PDB entries: Q96P65 (QRFP receptor) and 2RH1 (β 2 ‐adrenoceptor), resolution: 2.4 Å, sequence identity: 26%) (Neveu et al, ). TM and nonTM (upper: extracellular loop, down: intracellular loop) helices are shown as ribbons in red and orange respectively.…”

“…(B) LV‐2172 [cmpi 21 , aza‐β 3 ‐Hht 23 ]26RFa (19–26) (Neveu et al, ). (C) LV‐2185 [ADMA 25 ]26RFa (20–26) (Neveu et al, ).…”

“…Indeed, docking studies strongly suggest that the positively charged side chain of the Lys 19 residue rather than those of the Arg 25 moiety interacts with Glu 6.59 of QRFP receptors. Accordingly, in this pose, Cα of lysine‐19 is 8 Å far from that of Glu 6.59 , whereas the Cα‐Cα distance between Arg 25 and Glu 6.59 is >14 Å (Neveu et al, ).…”

“…Human QRFP receptor exhibits 26% sequence homology with the β 2 -adrenoceptor. Thus, a 3D molecular model of the human QRFP receptor has been built from the X-ray structure of the β 2 -adrenoceptor as a class-A receptor scaffold (Neveu et al, 2014). In addition to the seven helical TM domains, the human QRFP receptor model, like the β 2 -adrenoceptor template, displays one extracellular…”

“…Consistent with the widespread distribution of the receptor, 26RFa and QRFP have been found to regulate many physiological functions including energy homeostasis (Chartrel et al, ), bone formation (Baribault et al, ), hypothalamo‐pituitary‐gonadal activity (Navarro et al, ; Patel et al, ), insulin secretion (Egido et al, ; Granata et al, ; Prévost et al, ), locomotor activity (Do Rego et al, ) and analgesia (Yamamoto et al, ). The potential implication of these neuropeptides in various pathologies has prompted medicinal chemists to study the structure–activity relationships (SAR) of 26RFa in order to design selective agonists and antagonists (Le Marec et al, ; Neveu et al, , ; Georgsson et al, , ; Nordqvist et al, ).…”