TY - JOUR
T1 - Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis
AU - Gensure, Robert C.
AU - Carter, Percy H.
AU - Petroni, Brian D.
AU - Jüppner, Harald
AU - Gardella, Thomas J.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2001/11/16
Y1 - 2001/11/16
N2 - 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 (hP1RCAM-HR), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa 2,Ile5,Trp23,Tyr 36]PTHrP-(1-36)-amide (Bpa2-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-L-phenyl-alanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1RCAM-HR. This analog cross-linked to hP1RCAM-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 crosslink 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.
AB - 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 (hP1RCAM-HR), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa 2,Ile5,Trp23,Tyr 36]PTHrP-(1-36)-amide (Bpa2-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-L-phenyl-alanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1RCAM-HR. This analog cross-linked to hP1RCAM-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 crosslink 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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U2 - 10.1074/jbc.M106215200
DO - 10.1074/jbc.M106215200
M3 - Article
C2 - 11553625
AN - SCOPUS:0035900738
SN - 0021-9258
VL - 276
SP - 42692
EP - 42699
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -