Selective and nonselective inverse agonists for constitutively active type-1 parathyroid hormone receptors: evidence for altered receptor conformations.

ABSTRACT

The spontaneous signaling activity of some G protein-coupled receptors and the capacity of certain ligands (inverse agonists) to inhibit such constitutive activity are poorly understood phenomena. We investigated these processes for several analogs of PTH-related peptide (PTHrP) and the constitutively active human PTH/PTHrP receptors (hP1Rcs) hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist PTHrP(5-36) functioned as a weak partial/neutral agonist with both mutant receptors but was converted to an inverse agonist for both receptors by the combined substitution of Leu(11) and D-Trp(12). The N-terminally intact analog [Bpa(2)]PTHrP(1-36)-a partial agonist with the wild-type hP1Rc-was a selective inverse agonist, in that it depressed basal cAMP signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The ability of [Bpa(2)]PTHrP(1-36) to discriminate between the two receptor mutants suggested that H223R and T410P confer constitutive receptor activity by inducing distinct conformational changes. This hypothesis was confirmed by the observations that 1) the double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels that were 2-fold higher than those of either single mutant; and 2) hP1Rc-H223R and hP1Rc-T410P internalized (125)I-PTHrP(5-36) to markedly different extents. The overall results thus reveal that two different types of inverse agonists are possible for PTHrP ligands (nonselective and selective) and that constitutively active PTH-1 receptors can access different conformational states.