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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Feb;78(2):875–878. doi: 10.1073/pnas.78.2.875

Identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking

Gary L Johnson 1, Vincent I MacAndrew Jr 1, Paul F Pilch 1
PMCID: PMC319906  PMID: 6262779

Abstract

The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach 125I-labeled glucagon covalently to a rat liver membrane protein of Mr ≈ 53,000. Membranes that had been incubated with 125I-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 125I-labeled glucagon binding and covalent attachment to the Mr 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 125I-labeled glucagon to the Mr 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 125I-labeled glucagon to the Mr 53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of 125I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached 125I-labeled glucagon to the Mr 53,000 peptide with an efficiency of 1-2% can be used to attach 125I-labeled insulin covalently to a Mr 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.

Keywords: hydroxysuccinimidyl-p-azidobenzoate, covalent attachment, hormones

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Selected References

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