LOCALIZATION OF THE DOMAINS INVOLVED IN LIGAND-BINDING AND ACTIVATIONOF THE GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE RECEPTOR

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 demons trate complete specificity for their endogenous ligands, while utilizi ng similar second messenger pathways. In the current study chimeric GI P-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 I-125-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 p art 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 recepto r (IC50 = 1.33 +/- 0.19 nM; EC50 = 497.3 +/- 211.7 pM). Surprisingly c onstructs CH-2 and CH-3, while devoid of detectable I-125-GLP-1 bindin g, 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 recepto r activation is not completely dependent on, high affinity receptor bi nding. These data clearly demonstrate that the NT domain of the GTP re ceptor acts as the ligand-specific binding domain and that the first t ransmembrane domain is important for receptor activation.