Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling.
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.
Unson
Cecilia G
CG
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. unsonc@mail.rockefeller.edu
Wu
Cui-Rong
CR
Jiang
Youwei
Y
Yoo
Barney
B
Cheung
Connie
C
Sakmar
Thomas P
TP
Merrifield
R B
RB
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
D008024:Ligands; D010446:Peptide Fragments; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D005934:Glucagon