Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

• Published in: The Journal of biological chemistry Vol. 285; no. 40; pp. 30951 - 30958
• Main Authors: Prévost, Martine, Vertongen, Pascale, Raussens, Vincent, Roberts, David Jonathan, Cnudde, Johnny, Perret, Jason, Waelbroeck, Magali
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Adenylate Cyclase (Adenylyl Cyclase); Amino Acid Substitution; Biotinylation; Cell Line; Cell Surface Receptor; Cysteine; Cysteine - chemistry; Cysteine - genetics; Cysteine - metabolism; Data processing; G protein-coupled receptors; G Protein-coupled Receptors (GPCRs); Glucagon; Glucagon-like peptide 1 receptors; Humans; Models, Molecular; Molecular modelling; Mutagenesis; Mutation; Mutation, Missense; Protein Folding; Protein Structure and Folding; Protein Structure, Tertiary; Receptor mechanisms; Receptor Structure-Function; Receptors, Glucagon - chemistry; Receptors, Glucagon - genetics; Receptors, Glucagon - metabolism; Salts; Scanning; Tryptophan; Receptor Structure-Function; Cell Surface Receptor; G Protein-coupled Receptors (GPCRs); Protein Folding; Adenylate Cyclase (Adenylyl Cyclase)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.102814

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp63, Arg116, and Lys98 are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp63 and Arg116 form a salt bridge, whereas Lys98 is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.