Localization of the domains involved in ligand binding and activation of the glucose-dependent insulinotropic polypeptide receptor

• Published in: Endocrinology (Philadelphia) 1997-06, Vol.138 (6), p.2640-2643
• Main Authors: Gelling, R W, Wheeler, M B, Xue, J, Gyomorey, S, Nian, C, Pederson, R A, McIntosh, C H
• Format: Article
• Language: English
• Subjects: Adenylyl Cyclases - metabolism; Animals; Binding Sites; Binding, Competitive; CHO Cells; Cloning, Molecular; COS Cells; Cricetinae; Cyclic AMP - metabolism; Gastric Inhibitory Polypeptide - metabolism; Gastric Inhibitory Polypeptide - pharmacology; Glucagon - metabolism; Glucagon - pharmacology; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Kinetics; Models, Structural; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Protein Precursors - metabolism; Protein Precursors - pharmacology; Protein Structure, Secondary; Rats; Receptors, Gastrointestinal Hormone - biosynthesis; Receptors, Gastrointestinal Hormone - chemistry; Receptors, Gastrointestinal Hormone - metabolism; Receptors, Glucagon - biosynthesis; Receptors, Glucagon - chemistry; Receptors, Glucagon - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Signal Transduction; Transfection
• ISSN: 0013-7227
• DOI: 10.1210/en.138.6.2640

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.