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J. Biol. Chem., Vol. 276, Issue 46, 42692-42699, November 16, 2001

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Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis^*

Robert C. Gensure^§, Percy H. Carter, Brian D. Petroni, Harald Jüppner, and Thomas J. Gardella^¶

From the  Endocrine Unit and ^§ Pediatric Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R_CAM-HR), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa²,Ile⁵,Trp²³,Tyr³⁶]PTHrP-(1-36)-amide (Bpa²-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 to hP1R_CAM-HR at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro⁴¹⁵-Met⁴⁴¹, spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met⁴²⁵ to Leu converted Bpa²-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa²-containing analogs, as inverse agonism of Bpa²-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu¹¹,D-Trp¹²]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.

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