title: Localization of the domains involved in ligand binding and activation of the glucose-dependent insulinotropic polypeptide receptor.
abstract: The receptors for the two structurally related insulinotropic hormones Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-Like Peptide-1 (GLP-1) share approximately 40% sequence identity and demonstrate complete specificity for their endogenous ligands, while utilizing similar second messenger pathways. In the current study chimeric GIP-GLP-1 receptors were prepared, and the effect of domain-exchange on ligand binding and adenylyl cyclase activation examined. A chimera (CH-2) consisting of the first 132 amino acids of the external N-terminal (NT) domain bound 125I-GIP with high affinity (27.77 +/- 11.85 nM). However, for receptor coupling to cAMP production it was necessary to extend the NT into the first transmembrane (TM-1) region (CH-3: IC50 = 9.04 +/- 1.07 nM; EC50 = 17.1 +/- 3.5 nM). A chimera which included part of TM-3 (CH-4) demonstrated binding and signalling (IC50 = 8.33 +/- 0.14 nM; EC50 = 467.5 +/- 173.6 pM) similar to the wild type receptor (IC50 = 1.33 +/- 0.19 nM; EC50 = 497.9 +/- 211.7 pM). Surprisingly constructs CH-2 and CH-3, while devoid of detectable 125I-GLP-1 binding, were capable of eliciting GLP-1-specific cAMP production (EC50s CH-2 = 81.4 +/- 19.6 nM; CH-3 = 5.99 +/- 0.68 nM) suggesting that receptor activation is not completely dependent on high affinity receptor binding. These data clearly demonstrate that the NT domain of the GIP receptor acts as the ligand-specific binding domain and that the first transmembrane domain is important for receptor activation.
authors.affiliation: Department of Physiology, University of British Columbia, Vancouver, Canada.
authors.forename: R W
authors.initials: RW
authors.lastname: Gelling
authors.forename: M B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: J
authors.initials: J
authors.lastname: Xue
authors.forename: S
authors.initials: S
authors.lastname: Gyomorey
authors.forename: C
authors.initials: C
authors.lastname: Nian
authors.forename: R A
authors.initials: RA
authors.lastname: Pederson
authors.forename: C H
authors.initials: CH
authors.lastname: McIntosh
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005749:Gastric Inhibitory Polypeptide / Q000378:metabolism / Q000494:pharmacology*; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011498:Protein Precursors / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D011498:Protein Precursors; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D005749:Gastric Inhibitory Polypeptide; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; C041086:gastric inhibitory polypeptide receptor; D000242:Cyclic AMP; D000262:Adenylyl Cyclases