The DRY motif as a molecular switch of the human oxytocin receptor.
The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. Subsequently, both oxytocin and arginine vasopressin showed an increased potency in stimulating IP3 accumulation as compared to the wild-type receptor. Very interestingly, our experiments provide strong evidence that the D136(3.49)N mutant inhibits receptor signaling via Galpha(i)-mediated pathways while increasing the activity through the Galpha(q)-mediated pathways. Molecular simulations of the free and OT-bound forms of wild-type OTR and of the D136(3.49)N constitutively active mutant suggest that the receptor portions close to the E/DRY and NPxxY motifs are particularly susceptible to undergoing structural modification in response to activating mutations and agonist binding. Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.
Favre
Nicolas
N
Serono Pharmaceutical Research Institute, 14, Ch. des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.
Fanelli
Francesca
F
Missotten
Marc
M
Nichols
Anthony
A
Wilson
Janet
J
di Tiani
Mariastella
M
Rommel
Christian
C
Scheer
Alexander
A
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D019295:Computational Biology; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010121:Oxytocin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D018045:Receptors, Oxytocin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D018045:Receptors, Oxytocin; D014443:Tyrosine; D010121:Oxytocin; D001216:Asparagine; D001120:Arginine