Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties.

Incretins are endogenous peptides released from the gastrointestinal tract into the circulation during a meal that potentiate glucose-stimulated insulin secretion. At present, there are two established incretins: glucose-dependent insulinotropic polypeptide (GIP) and the truncated glucagon-like peptides (tGLPs), which are now being investigated for use in the treatment of diabetes mellitus. In the present study we cloned a rat islet GIP receptor complementary DNA (GIP-R1) to answer several important questions regarding the ligand-binding and intracellular signaling properties of the GP receptor. GIP-R1, when expressed transiently in monkey kidney (COS-7) or stably in Chinese hamster ovary (CHO-K1) cells, demonstrated comparable high affinity binding for either synthetic porcine (sp) GIP or synthetic human (sh) GIP. The IC50 values for displacement of 125IspGIP in CHO-K1 cells were 2.6 +/- 0.8 and 3.1 +/- 0.9 nM for two different preparations of shGIP, and 3.7 +/- 1.5 and 3.6 +/- 0.4 nM for two preparations of spGIP. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with apparent Kd values of 204 +/- 17 and 334 +/- 94 pM, respectively. Cells expressed 12-15 x 10(3) receptors/cell. In COS-7 cells, spGIP and shGIP also exhibited similar IC50 values (7.6 +/- 1.2 and 8.9 +/- 1.8 nM, respectively). The receptor in CHO-K1 cells bound GIP-(1-30) with lower affinity (IC50 = 39 +/- 17 nM), whereas the fragments GIP-(19-30), GIP-(18-28), and GIP-(21-26) showed no apparent binding. The specificity of the receptor was further examined using several structurally related peptides. Surprisingly, exendin-(9-39) Ex-(9-39), a GLP-1 receptor antagonist, and Ex-4-(1-39), a GLP-1 receptor agonist, demonstrated some affinity for the GIP receptor, with 39% and 21% displacement of 125IspGIP, respectively, at 1 microM. Other members of the secretin/vasoactive intestinal peptide family of peptides tested showed no interaction. GIP-R1 receptor binding correlated with activation of the adenylyl cyclase system, whereby spGIP and shGIP evoked concentration-dependent increases in cAMP accumulation with EC50 values of 8.7 +/- 1.5 x 10(-10)M and 8.1 +/- 1.6 x 10(-10)M for spGIP and shGIP, respectively. Increases in cAMP in the presence of 10 nM spGIP were not dependent on the ambient glucose concentration, with 22- and 18-fold increases in cAMP accumulation at 0.1 and 5.5 mM glucose, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)