Activation Mechanism of Human Oxytocin Receptor: A Combined Study of Experimental and Computer-Simulated Mutagenesis

Abstract

The aim of this study was to investigate the molecular changes associated with the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartate-arginine-tyrosine motif located in the second intracellular domain gave rise to the first known constitutively active oxytocin receptor (R137A), whereas mutation of the aspartic acid located in the second transmembrane domain led to an inactive receptor (D85A). The structural features of the constitutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest that, although differently triggered, the activation process induced by the agonist and the activating mutation are characterized by the opening of a solvent exposed site formed by the 2nd intracellular loop, the cytosolic extension of helix 5, and the 3rd intracellular loop; on the contrary, the D85A mutation prevents oxytocin from triggering the opening of a cytosolic site. On the basis of these findings, we hypothesize that this cytosolic crevice plays an important role in G protein recognition. Finally, comparative analysis of the free- and agonist-bound forms of the wild-type oxytocin receptor and alpha1B adrenergic receptor suggests that the highly conserved polar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.

Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions

“…The cluster includes critical GPCR motifs and residues, such as DRY motif in TM3, NPXXY motif in TM7, Cys-110 in TM3 (disulfide-bonded), and Asn-55 in TM1. Most of its trace residues belong to TM2, TM3, TM7, and to a smaller extent TM6, consistent with mutagenesis, spectroscopy, and cross-linking evidence that the latter three helices undergo rigid-body motions upon activation (41)(42)(43)(44)(45).…”

Evolutionary pattern in the OXT-OXTR system in primates: Coevolution and positive selection footprints

“…Here we obtained consistent evidence for positive selection in OXT for the Cebidae branch (OXT-8Pro and OXT3Val-8Pro). For OXTR, we identified sites that seem to be relevant for the function of the OXT-OXTR system beyond those previously identified (25)(26)(27)(28). For three of them (sites 6, 251, and 255), signals of positive selection were detected.…”

“…Other important results emerged regarding OXTR binding sites (Arg-34, Phe-103, Tyr-209, and Phe-284) (25)(26)(27)(28). These sites are conserved in the species sampled, with the exception of S. sciureus, which showed the Phe103Tyr change (7).…”

“…Coevolution was tested at the intramolecular (within single molecules or genes) or intermolecular (between different molecules or genes) levels (47). First we considered recognized specific binding sites, OXT amino acid chain positions 3 and 8, and OXTR amino acid chain positions 34, 103, 209, and 284 (25)(26)(27)(28). Second, we examined coevolutionary processes through the Bayesian Spidermonkey tool (48), available at the Datamonkey server (www.…”

Neurohormones: oxytocin, vasopressin and related peptides – structure, genes, receptors, and evolution

“…Several regulatory elements of interest were identified, including cyclic adenosine monophosphate (cAMP) response element (CRE), a serum response element (SRE), several activator protein-1 elements (AR-1) in addition to a palindromic oestrogen response element (ERE). In transfection assays, protein kinase C (PKC) and protein kinase dependent on cAMP (PKA) activation by phorbol ester and forskolin treatment optimally enhanced the transcription of the OTR (Fanelli et al, 1999;Telgmann et al, 2003).…”