The biochemical characterization of the native pancreatic cholecystokinin receptor using affinity labeling approaches

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Abstract

Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using 'intrinsic' probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a 'topographical mapping' probe, which incorporates a flexible spacer of variable length between a CCK- like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other. In addition to the identification of proteins which represent the hormone- binding protein and proteins associated with it, affinity-labeling approaches can provide structural information about post-translational modifications of these proteins, which clearly complement and extend the primary amino acid sequence data now made available by receptor cDNA cloning. There are data to support the glycosylation and phosphorylation of the rat pancreatic CCK receptor. In addition, it is possible to use photoaffinity labeling with 'intrinsic' probes to label directly domains and residues in the receptor which are critical to agonist binding.

Original languageEnglish (US)
Pages (from-to)441-448
Number of pages8
JournalYale Journal of Biology and Medicine
Volume65
Issue number5
StatePublished - 1992

Fingerprint

Cholecystokinin Receptors
Labeling
Cholecystokinin
Ligands
Carrier Proteins
Cell Membrane
Proteins
Peptide Mapping
Acinar Cells
Membrane Glycoproteins
Post Translational Protein Processing
Glycosylation
Cell membranes
Organism Cloning
Amino Acid Sequence
Membrane Proteins
Peptide Hydrolases
Complementary DNA
Rats
Phosphorylation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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abstract = "Affinity labeling has been a powerful tool for the biochemical characterization of sparse molecules which bind to a ligand probe in a specific, high-affinity manner. The rat pancreatic acinar cell receptor for cholecystokinin (CCK), the major physiologic hormonal stimulant of pancreatic exocrine secretion, has been the target of such investigation. Of interest, affinity-labeling studies have identified two distinct plasma membrane glycoproteins as candidates to represent this receptor. The initial candidate, which was identified using 125I-Bolton Hunter-labeled CCK-33 as probe, migrates on a SDS-polyacrylamide gel as a broad band in the M(r) = 80,000 range. Subsequently, using shorter probes in which the site of covalent attachment was closer to the receptor-binding domain of the probe, a band of M(r) = 85,000-95,000 was specifically labeled. Deglycosylation and protease-peptide mapping demonstrated that these bands represent distinct molecules. Using 'intrinsic' probes of the receptor, in which a photolabile residue was sited within the pharmacophoric domain of the ligand, attention was focused on the latter candidate as representing the binding protein. Insight into the relationship between these proteins as they reside in the plasma membrane was contributed by labeling with a 'topographical mapping' probe, which incorporates a flexible spacer of variable length between a CCK- like ligand and a photolabile residue. This procedure confirmed that these two minor membrane proteins are spatially associated with each other. In addition to the identification of proteins which represent the hormone- binding protein and proteins associated with it, affinity-labeling approaches can provide structural information about post-translational modifications of these proteins, which clearly complement and extend the primary amino acid sequence data now made available by receptor cDNA cloning. There are data to support the glycosylation and phosphorylation of the rat pancreatic CCK receptor. In addition, it is possible to use photoaffinity labeling with 'intrinsic' probes to label directly domains and residues in the receptor which are critical to agonist binding.",
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