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Vol. 286, Issue 2, 593-598, August 1998
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
(Ki = 0.5 ± 0.1 nM) and 99% in the low affinity state (Ki = 23 ± 3 nM).
Mutant receptor binding best fit a single affinity state, having values
for Ki 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 Gs or
increased aberrant coupling with Gi, 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 Gs, thereby
resulting in inverse agonism.
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