title: Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling.
abstract: To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.
authors.affiliation: Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. unsonc@mail.rockefeller.edu
authors.forename: Cecilia G
authors.initials: CG
authors.lastname: Unson
authors.forename: Cui-Rong
authors.initials: CR
authors.lastname: Wu
authors.forename: Youwei
authors.initials: Y
authors.lastname: Jiang
authors.forename: Barney
authors.initials: B
authors.lastname: Yoo
authors.forename: Connie
authors.initials: C
authors.lastname: Cheung
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: R B
authors.initials: RB
authors.lastname: Merrifield
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D019556:COS Cells; D005110:Extracellular Space / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D005934:Glucagon