Vol. 286, Issue 2, 593-598, August 1998

Protean Effects of a Natural Peptide Agonist of the G Protein-Coupled Secretin Receptor Demonstrated by Receptor Mutagenesis¹

Subhas C. Ganguli, Chan-Guk Park, Martin H. Holtmann, Elizabeth M. Hadac, Terry P. Kenakin and Laurence J. Miller

Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota (S.C.G., C.-G.P., M.H.H., E.M.H., L.J.M.) and Department of Receptor Biochemistry, Glaxo Wellcome Inc, Research Triangle Park, North Carolina (T.P.K.)

G protein-coupled receptors initiate signaling cascades after associating with heterotrimeric G proteins. This is typically initiated by agonist binding, but can also occur spontaneously, particularly in receptors bearing distinct missense mutations. Two such mutations in the parathyroid hormone receptor are associated with constitutive activity, manifesting clinically as Jansen's metaphyseal chondroplasia. We introduce analogous mutations separately and together into the secretin receptor to explore their impact on another family member. Constructs were expressed transiently in COS cells, and had binding and signaling (cAMP generation) studied. Each construct was processed appropriately to lead to cell surface expression and signaling. Secretin bound to the wild-type receptor with two affinity states recognized, 1% of sites in the high affinity state (K_i = 0.5 ± 0.1 nM) and 99% in the low affinity state (K_i = 23 ± 3 nM). Mutant receptor binding best fit a single affinity state, having values for K_i of 5 ± 1 nM (H156R), 8 ± 1 nM (T322P) and 6 ± 1 nM (H156R/T322P), with each of these demonstrating a shift to higher affinity than the predominent low affinity state of the wild-type receptor. Each mutant receptor expressed small to moderate constitutive activity, with basal levels of cAMP activity greater than control (P < .01): H156R, 1.4-fold; T322P, 4.5-fold and H156R/T322P, 6.8-fold. The level of basal activity of even the most active construct was only 15% of the maximal response of wild-type receptor. Although each of the single site mutants responded to secretin by increasing their cAMP levels in a concentration-dependent manner, the dual mutant decreased its cAMP in response to hormone (EC₅₀ = 13 nM). Thus, a natural agonist had become an inverse agonist at this unique construct. Because this could reflect reduced normal coupling with G_s or increased aberrant coupling with G_i, the mechanism was further explored using pertussis toxin and a stable analogue of GTP. Although ligand-binding determinants were retained in the dual receptor mutant, the conformation of this receptor upon secretin binding effected a reduction in its basal coupling with G_s, thereby resulting in inverse agonism.