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Structure-function studies of the gastric inhibitory polypeptide/glucose dependent insulinotropic polypeptide (GIP) receptor Gelling, Richard Wayne


Incretins are hormones released from the gastrointestinal tract into the circulation during and after a meal that potentiate glucose-stimulated insulin secretion. Glucose dependent insulinotropic polypeptide (GIP) is now accepted as the most important incretin and, along with the glucagon-like peptide 1 (GLP-1 (7-36) amide) has therapeutic potential in Non-Insulin Dependent Diabetes Mellitus (NDDDM). In the present study, a rat islet GIP receptor complementary (c)DNA (GIP-R1) was cloned and characterized. The islet cDNA was identical to that previously identified in a tumor cell line, except for a single nucleotide polymorphism resulting in one amino acid difference (Glu21→ Gln21). When expressed transiently in COS-7 cells or stably in CHO-K1 cells the receptor displayed specific high affinity ¹²⁵I-GIP binding in both saturation (200-300 pM) and competition (IC₅₀ 1-8 nM) binding studies, and GIPdependent increases in cAMP production (EC₅₀ 0.069-0.70 nM). Cells expressing GIP-R1 exhibited equivalent signaling in response to porcine and human GIP. In addition, COS-7 cells expressing the GIP-R1 cDNA displayed a biphasic increase in intracellular calcium in response to GIP. Structure-function studies of GIP showed that the peptide could be truncated at its carboxy-terminal at residue 30 (GIP 1-30 amide) without affecting receptor affinity or efficacy. In contrast, amino-terminal truncation of GIP 1-30 resulted in fragments with reduced affinity and lacking receptor activation activity, that antagonized GIP-stimulated cAMP production. Importantly, GIP 6-30amide bound with nearly identical affinity to GIP but was a potent inhibitor of GIP action in vitro, suggesting that this region contains the binding core and that amino-terminal residues are important for receptor activation. The latter finding is important given that GTP is metabolized by dipeptidyl-peptidase (DP) IV to biologically inactive GTP 3-42. The analogs Ppa¹-GIP 1-30 and D-Ala²-GTP 1- 30amide were shown to be resistant to DP IV degradation in vitro, but had slightly reduced affinity and efficacy at the GTP receptor. Such DP IV resistant analogs may be useful in NIDDM treatment. Oligonucleotide-directed mutagenesis was used to examine regions important for ligand binding, receptor activation, and G-protein coupling. Studies of GTP/GLP-1 receptor chimeras indicated that the high affinity GIP binding domain lies within the extracellular amino-terminal of the GIP receptor, while the first transmembrane domain appears critical for GTP-specific receptor activation. A similar region of the GLP-1 receptor may be important for GLP-1 receptor activation. The effect of truncating the carboxy-terminal-tail of the GIP receptor on ligand binding, second messenger coupling, and internalization was examined. Truncation by >37 amino acids greatly decreased expression, and a minimum carboxy-terminal tail length of 13 amino acids appears to be required for receptor expression. In contrast the carboxy-terminal-tail could be truncated by up to 50 amino acids without affecting receptor affinity, and with only small effects on G-protein coupling and receptor internalization. These are the first detailed structure-function studies on GIP and its receptor in a cell system uncomplicated by factors inherent in whole animal preparations or immortalized P-cell lines. Further studies may lead to a better understanding of the apparent reduction of GTP receptors in NIDDM, and the development of GIP analogs that are useful in its treatment.

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