Localization of the Domains Involved in Ligand Binding and Activation of the Glucose-Dependent Insulinotropic Polypeptide Receptor
0303 health sciences
Binding Sites
CHO Cells
Gastric Inhibitory Polypeptide
Glucagon
Binding, Competitive
Glucagon-Like Peptide-1 Receptor
Peptide Fragments
Protein Structure, Secondary
Rats
Models, Structural
Kinetics
03 medical and health sciences
Glucagon-Like Peptide 1
Cricetinae
COS Cells
Cyclic AMP
Animals
Humans
Cloning, Molecular
Protein Precursors
Adenylyl Cyclases
DOI:
10.1210/endo.138.6.9104
Publication Date:
2014-01-08T11:09:05Z
AUTHORS (7)
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.
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