title: Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.
abstract: 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, [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 to 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 cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-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 Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism 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 extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.
authors.affiliation: Endocrine Unit and Pediatric Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: R C
authors.initials: RC
authors.lastname: Gensure
authors.forename: P H
authors.initials: PH
authors.lastname: Carter
authors.forename: B D
authors.initials: BD
authors.lastname: Petroni
authors.forename: H
authors.initials: H
authors.lastname: Jüppner
authors.forename: T J
authors.initials: TJ
authors.lastname: Gardella
mesh_terms: D000818:Animals; D019556:COS Cells; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020128:Inhibitory Concentration 50; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010281:Parathyroid Hormone / Q000819:agonists* / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000819:agonists* / Q000737:chemistry*; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D010281:Parathyroid Hormone; D010455:Peptides; D018016:Receptors, Parathyroid Hormone; D008715:Methionine; D000242:Cyclic AMP; D007930:Leucine