Phosphatidylinositol-specific phospholipase C (PLC) plays an important role in the regulation of insulin secretion by diverse nutrient and hormone secretagogues. We have provided insights into the pathways that mediate glucose stimulation of PLC; however, the exact signaling mechanisms and the specific isozymes involved are not known. In an effort to determine the PLC isozymes that are activated by glucose, we have identified a novel PLC that is homologous to the Ras/Rap l-binding PLC210 recently cloned in Caenorhabditis elegans. This PLC represents a new class whose regulation and function are not known. Evidence in the literature suggests that glucose may signal through Ras and Rap1 and that Ras stimulates PLC activity in a beta cell line. It is our hypothesis that in the beta cell, glucose and hormones couple to this novel PLC through Ras and/or Rap1. The long-term objective of this proposal, therefore, is to use current molecular, biochemical, and physiologic approaches to define the regulation of this novel PLC and determine its role in the pancreatic beta cell.
Our specific aims are threefold: 1) We will clone this PLC by PCR and hybridization based techniques, purify it as an expressed GST fusion protein in E. coli, and characterize its catalytic activity. In addition, we will determine its species and tissue dependent expression by Western and Northern blot analysis. 2) We will characterize the regulation of this PLC by G-proteins by examining the interaction of its C-terminus with Ras, Rap1 and other specific G-proteins, in vitro using a protein-protein binding assay, and in vivo using a yeast two-hybrid assay. We will also determine which G-proteins regulate its enzymatic activity in a reconstitution assay and in co-expression studies in Cos-7 cells. 3) We will elucidate the regulation and role of this enzyme in the beta cell, first by determining which secretagogues stimulate Ras and Rap1 using a specific effector binding assay; second by determining the effect of overexpressing these and other G-proteins on endogenous PLC activity; and finally, by determining the effect of overexpressing this PLC on secretagogue stimulation of PLC activity and insulin secretion. This novel signaling pathway may play a critical role in glucose and hormone stimulated insulin secretion and beta cell function.
