Activation mechanism of human oxytocin receptor: A combined study of experimental and computer-simulated mutagenesis

The aim of this study was to investigate the molecular changes associated w ith the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartat e-arginine-tyrosine motif located in the second intracellular domain gave r ise to the first known constitutively active oxytocin receptor (R137A), whe reas mutation of the aspartic acid located in the second transmembrane doma in led to an inactive receptor (D85A). The structural features of the const itutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest tha t, although differently triggered, the activation process induced by the ag onist and the activating mutation are characterized by the opening of a sol vent exposed site formed by the 2nd intracellular loop, the cytosolic exten sion 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 ana lysis of the free- and agonist-bound forms of the wild-type oxytocin recept or and alpha(1B) adrenergic receptor suggests that the highly conserved pol ar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.