Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus
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Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus. / Runge, Steffen; Gram, Christian; Bräuner-Osborne, Hans; Madsen, Kjeld; Knudsen, Lotte B; Wulff, Birgitte S.
In: Journal of Biological Chemistry, Vol. 278, No. 30, 25.07.2003, p. 28005-10.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus
AU - Runge, Steffen
AU - Gram, Christian
AU - Bräuner-Osborne, Hans
AU - Madsen, Kjeld
AU - Knudsen, Lotte B
AU - Wulff, Birgitte S
PY - 2003/7/25
Y1 - 2003/7/25
N2 - The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.
AB - The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.
KW - Amino Acid Sequence
KW - Aspartic Acid
KW - Cell Line
KW - DNA, Complementary
KW - Dose-Response Relationship, Drug
KW - Epitopes
KW - Genes, Reporter
KW - Glucagon
KW - Glutamic Acid
KW - Humans
KW - Inhibitory Concentration 50
KW - Models, Biological
KW - Molecular Sequence Data
KW - Point Mutation
KW - Protein Binding
KW - Protein Structure, Tertiary
KW - Receptors, Glucagon
KW - Sequence Homology, Amino Acid
U2 - 10.1074/jbc.M301085200
DO - 10.1074/jbc.M301085200
M3 - Journal article
C2 - 12724331
VL - 278
SP - 28005
EP - 28010
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -
ID: 45596711