UBC Theses and Dissertations
Adipose tissue expression, alternative splicing, desensitization and internalization of the glucose-dependent insulinotropic polypeptide receptor Gill, Ruth Ellen
Glucose dependent insulinotropic polypeptide (GIP) is a gut hormone which plays an important role as an incretin. Thus, the GIP receptor, a member of the G-protein coupled receptor (GPCR) family, is expressed in the β-cells of the endocrine pancreas. However, it is also expressed in other tissues, including adipose tissue, where GIP exerts effects on fat metabolism. Non-insulin dependent diabetes mellitus (NIDDM), a disease characterized by impaired insulin secretion and insulin resistance, is associated with obesity. Therefore, the possibility of altered GIP receptor expression was examined in the Zucker fatty (fa/fa) rat, a model of obesity. In contrast to previous studies, 11 week old obese Zucker rats did not show higher plasma glucose concentrations in response to oral glucose than did their lean littermates, but this is probably due to differences in statistical analysis. However, the 16 week old obese animals showed fasting hyperglycemia, severe glucose intolerance and greater plasma GIP responses to oral glucose than did their lean littermates, suggesting that they had become diabetic. The 11 week old lean and obese Zucker rats displayed nearly identical levels of adipose tissue GIP receptor mRNA, therefore, the adipose cell GIP receptor expression appears not to be involved in the phenotype of the animal at this stage of development. The GIP receptor is a member of the secretin/VIP subfamily of GPCRs, several of whose members exist in alternatively spliced forms. The presence of the GIP receptor in tissues other than the endocrine pancreas suggests the possibility of different signal transduction pathways. The third intracellular (i3) loop of many GPCRs is important for G-protein recognition and coupling, so this area of the GIP receptor was examined for alternative splicing. An alternatively spliced variant of the GIP receptor expressed predominantly in islet tissue was detected and predicted to encode a truncated form of the GIP receptor. A second splice variant expressed predominantly in adipose tissue was predicted to encode a 28 amino acid insert located at the C-terminal end of the i3 loop. The second and third intracellular loops of the GIP receptor contain several serine and threonine residues. As potential phosphorylation sites, these residues were examined for their ability to mediate receptor desensitization and internalization. The mutation of Ser-248, Ser-320, Thr-324 and Ser-339 to alanines resulted in a reduced ability to stimulate cyclic-adenosine monophosphate (cAMP) production in response to GIP stimulation as compared to the wild type GIP receptor (wtGIPR). The mutation of Thr- 342 to alanine resulted in an increased ability to stimulate cAMP production in response to GIP stimulation in low expressing subclones. The wild type GIP receptor did not show significant desensitization in response to preincubation with GIP, so it was not possible to determine the result of mutations on receptor desensitization. GIPR/S248A was the only mutant receptor to show a decrease in internalization rate and maximum internalization as compared to the wtGIPR. Neither GIPR/S320A nor GIPR/T324A showed changes in internalization rate or maximum internalization compared to the wtGIPR, while GIPR/S339A and GIPR/T342A showed an increase in both these parameters. Therefore, Ser-339 and Thr-342 may act as negative regulators of receptor internalization. The above studies have added to the growing knowledge about GIP's signaling pathways, its role in adipose tissue and its potential role in obesity and NIDDM.
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