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, Thomas J. Gardella

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 (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.

Original languageEnglish (US)
Pages (from-to)42692-42699
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number46
DOIs
StatePublished - Nov 16 2001
Externally publishedYes

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Parathyroid Hormone Receptor Type 1
Parathyroid Hormone
Parathyroid Hormone-Related Protein
Alanine
Substitution reactions
Enchondromatosis
Ligands
Valine
Amides
Peptides
Mutation
parathyroid hormone-related peptide (1-36)

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis. / Gensure, Robert C.; Carter, Percy H.; Petroni, Brian D.; Jüppner, Harald; Gardella, Thomas J.

In: Journal of Biological Chemistry, Vol. 276, No. 46, 16.11.2001, p. 42692-42699.

Research output: Contribution to journalArticle

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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 (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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AU - Gardella, Thomas J.

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