Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis

We have investigated receptor structural components responsible for ligand- dependent inverse agonism in a constitutively active mutant of the human pa rathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) recepto r type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was origi nally identified in Jansen's chondrodysplasia and is altered in transmembra ne domain (TM) 2. We utilized the PTHrP analog, [Bpa(2), Ile(5),Trp(23),Tyr (36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP- (1-36)), which has valine 2 replaced by p-benzoyl-L-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked t o hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second crosslink si te (site B) was within the TM4/TM5 region. Within the site A interval, subs titution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse a gonist to a weak partial agonist; this conversion was accompanied by a rela tive shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agon ism of [Leu(11), D-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extrace llular end of TM6 of the hP1R play an important role in modulating the conv ersion between active and inactive receptor states.