Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1-36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking

Robert C. Gensure, Thomas J. Gardella, Harald Jüppner

Research output: Contribution to journalArticle

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Abstract

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [I5,W23,Y36]PTHrP-(1-36)-amide with individual p-benzoyl-L-phenylalanine (Bpa) substitutions at positions 22-35. When tested with LLC-PK1 cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC50 of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa 24-substituted analog, similar to that of the parent compound. The radiolabeled Bpa23-, Bpa27-, Bpa28-, and Bpa33-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I5,Bpa23,Y36]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I 5,W23,Bpa27,Y36]PTHrP-(1-36)-amide cross-linked to residues 96-102; [I5,W23,Bpa 28,Y36]PTHrP-(1-36)-amide cross-linked to residues 64-95; and [I5,W23, Bpa33,Y 36]PTHrP-(1-36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

Original languageEnglish (US)
Pages (from-to)28650-28658
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number31
DOIs
StatePublished - Aug 3 2001
Externally publishedYes

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Parathyroid Hormone Receptor Type 1
Parathyroid Hormone-Related Protein
Parathyroid Hormone
Amides
Phenylalanine
LLC-PK1 Cells
Substitution reactions
Hormones
Ligands
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1-36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking. / Gensure, Robert C.; Gardella, Thomas J.; Jüppner, Harald.

In: Journal of Biological Chemistry, Vol. 276, No. 31, 03.08.2001, p. 28650-28658.

Research output: Contribution to journalArticle

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title = "Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1-36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking",
abstract = "The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [I5,W23,Y36]PTHrP-(1-36)-amide with individual p-benzoyl-L-phenylalanine (Bpa) substitutions at positions 22-35. When tested with LLC-PK1 cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC50 of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa 24-substituted analog, similar to that of the parent compound. The radiolabeled Bpa23-, Bpa27-, Bpa28-, and Bpa33-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I5,Bpa23,Y36]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I 5,W23,Bpa27,Y36]PTHrP-(1-36)-amide cross-linked to residues 96-102; [I5,W23,Bpa 28,Y36]PTHrP-(1-36)-amide cross-linked to residues 64-95; and [I5,W23, Bpa33,Y 36]PTHrP-(1-36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.",
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N2 - The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [I5,W23,Y36]PTHrP-(1-36)-amide with individual p-benzoyl-L-phenylalanine (Bpa) substitutions at positions 22-35. When tested with LLC-PK1 cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC50 of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa 24-substituted analog, similar to that of the parent compound. The radiolabeled Bpa23-, Bpa27-, Bpa28-, and Bpa33-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I5,Bpa23,Y36]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I 5,W23,Bpa27,Y36]PTHrP-(1-36)-amide cross-linked to residues 96-102; [I5,W23,Bpa 28,Y36]PTHrP-(1-36)-amide cross-linked to residues 64-95; and [I5,W23, Bpa33,Y 36]PTHrP-(1-36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

AB - The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [I5,W23,Y36]PTHrP-(1-36)-amide with individual p-benzoyl-L-phenylalanine (Bpa) substitutions at positions 22-35. When tested with LLC-PK1 cells stably transfected with human P1R (hP1R), the apparent binding affinity and the EC50 of agonist-stimulated cAMP accumulation for each analog was, with the exception of the Bpa 24-substituted analog, similar to that of the parent compound. The radiolabeled Bpa23-, Bpa27-, Bpa28-, and Bpa33-substituted compounds affinity-labeled the hP1R sufficiently well to permit subsequent mapping of the cross-linked receptor region. Each of these peptides cross-linked to the amino-terminal extracellular domain of the P1R: [I5,Bpa23,Y36]PTHrP-(1-36)-amide cross-linked to the extreme end of this domain (residues 33-63); [I 5,W23,Bpa27,Y36]PTHrP-(1-36)-amide cross-linked to residues 96-102; [I5,W23,Bpa 28,Y36]PTHrP-(1-36)-amide cross-linked to residues 64-95; and [I5,W23, Bpa33,Y 36]PTHrP-(1-36)-amide cross-linked to residues 151-172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex.

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