The effects of vasoactive intestinal peptide (VIP) on intestinal secretion have been well characterized. Clinically, tumors producing VIP are responsible for the syndrome of watery diarrhea, hypokalemia and gastric achlorhydria described by Verner and Morrison. However, the interaction between VIP and its receptor on the target cell, the first step responsible for specific signal transduction, has only recently been examined. With the groundwork laid by our previous studies on the structural on the intestinal VIP receptor, we plan to: 1. Raise monoclonal and polyclonal antibodies against the VIP receptor using the purest receptor preparation available (see below). 2. Purify the VIP receptor relying mainly on gel filtration and affinity chromatography (using either VIP or antibodies against the receptor). If required, reconstitute the purified receptor into liposomes to define its function. 3. Using different endoglycosidases, study the degree and type of glycosylation of the VIP receptor located on the ER-Golgi and laterobasal membranes of the enterocyte. With the panel of monoclonal antibodies described in 1, attempt to correlate a function or structure to each domain of the receptor. 4. Study the synthesis, glycosylation, vectorial processing and transport of the metabolically radiolabeled VIP receptor in pulse chase experiments using the isolated intestinal loop. 5. With the intestinal loop or the human colonic adenocardinoma cell line, HT 29, study the cellular cycle of the receptor (internalization, degradation, recycling).
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