Photoaffinity Labeling Demonstrates Physical Contact between Vasoactive Intestinal Peptide and the N-terminal Ectodomain of the Human VPAC1 Receptor

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at...

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Veröffentlicht in:The Journal of biological chemistry 2003-09, Vol.278 (38), p.36531-36536
Hauptverfasser: Tan, Yossan-Var, Couvineau, Alain, Van Rampelbergh, Jean, Laburthe, Marc
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container_issue 38
container_start_page 36531
container_title The Journal of biological chemistry
container_volume 278
creator Tan, Yossan-Var
Couvineau, Alain
Van Rampelbergh, Jean
Laburthe, Marc
description Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67–137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109–120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137–153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.
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To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67–137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109–120 fragment (GWTHLEPGPYPI). 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To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67–137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109–120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137–153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>12807902</pmid><doi>10.1074/jbc.M304770200</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
Animals
Binding, Competitive
CHO Cells
Cricetinae
Cyanogen Bromide - pharmacology
Electrophoresis, Polyacrylamide Gel
Glycosylation
Humans
Kinetics
Ligands
Light
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Peptides - chemistry
Photoaffinity Labels - pharmacology
Protein Binding
Protein Structure, Tertiary
Receptors, Vasoactive Intestinal Peptide - chemistry
Receptors, Vasoactive Intestinal Polypeptide, Type I
Recombinant Fusion Proteins - metabolism
Transfection
Vasoactive Intestinal Peptide - chemistry
title Photoaffinity Labeling Demonstrates Physical Contact between Vasoactive Intestinal Peptide and the N-terminal Ectodomain of the Human VPAC1 Receptor
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