Identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking.
The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach (125)I-labeled glucagon covalently to a rat liver membrane protein of M(r) approximately 53,000. Membranes that had been incubated with (125)I-labeled glucagon were treated in the dark with hydroxysuccinimidyl-p-azidobenzoate, and a covalent complex was then formed by irradiation with ultraviolet light. Characteristics of (125)I-labeled glucagon binding and covalent attachment to the M(r) 53,000 peptide were consistent with this peptide being a component of the glucagon receptor involved in the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. Binding and covalent attachment of (125)I-labeled glucagon to the M(r) 53,000 peptide were inhibited by glucagon concentrations that were within the dose-response curve for adenylate cyclase activation, and GTP specifically decreased the photoaffinity crosslinking of (125)I-labeled glucagon to the M(r) 53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of (125)I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached (125)I-labeled glucagon to the M(r) 53,000 peptide with an efficiency of 1-2% can be used to attach (125)I-labeled insulin covalently to a M(r) 125,000 peptide with an efficiency of approximately 10%. This peptide has been shown to be a subunit of the high-affinity insulin-binding site in rat liver membranes. The technique of photoaffinity crosslinking with agents like hydroxysuccinimidyl-p-azidobenzoate provides a rapid, simple method of covalently attaching ligands to their putative receptors. Photoaffinity crosslinking does not require chemical modification of the labeled ligand and has a less stringent requirement for specific reactive groups than the commonly used bifunctional crosslinking reagents.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D005934:Glucagon / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018027:Receptors, Glucagon
D001386:Azides; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D018027:Receptors, Glucagon; C029534:hydroxysuccinimidyl-4-azidobenzoate; D005934:Glucagon; D000262:Adenylyl Cyclases