The overall objective of this proposal is to examine the factors which regulate the expression and cellular sorting of the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR) in developing rat intestine and polarized epithelial cells. Quantitative Western blot analysis and Northern blot analysis will characterize and quantify the protein and mRNA levels, respectively, of the IGF-II/CI-MPR and CD-MPR. In addition, IGF-II binding studies will be performed to determine the functional activity of the IGF-II/CI-MPR in intestinal membranes. These studies will be carried out using a) different anatomical regions of the intestine, b) villus and crypt cells, and c) intestine from different developmental stages of the rat. Similar studies as above will be carried out on a human colon adenocarcinoma cell line, Caco-2, which spontaneously undergoes enterocyte-like differentiation in culture. The biosynthesis and processing of the MPRs will be characterized in these cells as will the effect IGF-II and other growth factors have on this cell's rate of differentiation. The cell surface distribution of the MPRs in polarized cells grown on filters will be determined by cell surface labeling techniques and uptake studies using 125I-IGF-II and [3H]lysosomal enzymes. In addition, the kinetics of intracellular movement of the MPRs will be analyzed in these cells. The role that the MPRs' covalent modifications (i.e., acylation, phosphorylation, glycosylation) have as regulators of the receptors' intracellular targeting will be addressed using metabolic labeling studies in conjunction with processing inhibitors as well as site-directed mutagenesis of the MPRs' cDNA. Once the targeting pathway of the MPRs has been established, the structural elements of the receptors' cytoplasmic domain and/or extracytoplasmic domain important for their intracellular targeting will be examined by site-directed mutagenesis of the MPRs' cDNA. These mutant MPR cDNAs will be transfected into Caco-2 and/or opossum kidney cells in order to study the effect these mutations have on the intracellular targeting of the MPRs in a polarized cell. the studies outlined in this proposal will examine at the cellular and molecular levels the possible roles the MPRs have in epithelial differentiation and in regulating the functional capacity of enterocytic lysosomes, and thus will provide a basis for a clearer understanding of the role of this organelle in protein metabolism in the gut.
