The transport system involving intrinsic factor (IF) and its receptor (IF-Rec) is only one of a few well characterized receptor-mediated systems in mammalian intestine whereby ligands can be absorbed into the enterocyte and eventually into the blood. Similar receptor-mediated transport systems involve the other cobalamin (cbl) binding proteins, including R protein or haptocorrin (hepatocyte) and transcobalamin II or TCII (all somatic cells). This grant will study the physiology and structural interactions of these transport systems, with particular emphasis on IF. We have isolated CDNA clones encoding the entire structural regions of rat and human IF and of hog R protein, and genomic clones that identify all the axons of human IF with the exception of the first 30 bp of the 5' UT portion of the CDNA. We have expressed rat IF in a eukaryotic system (COS-1 cells), and found that it can be active in the absence of N-glycosylation. Assays are available to follow the binding of cbl to IF, and of IF-cbl to brush border membranes, using IF produced either by transfection or in a cell-free system. IF and haptocorrin have been found immunologically to coexist in cells of foregut tissues, i.e. the gastric mucosa, and in ductal cells of the salivary glands and pancreas. We have also identified a haptocorrin receptor in intestine, corresponding with the minor 49-54 Kda subunits of the hepatic asialoglycoprotein receptor (ASGP-R). Human TCII has been cloned by other workers. We propose to produce radiolabelled IF in stably transfected cells to use for metabolic studies, to identify the cbl and receptor binding regions of IF, to study the fate of labelled IF in vivo in the rat and in vitro using Caco-2 cells, to study the secretory pathway of IF and haptocorrin in gastric parenchymal cells, and in salivary and pancreatic ductal cells, to isolate and study further the importance of the intestinal ASGP-R, and to isolate a TCII clone to transfect Caco-2 cells, to test an hypothesis that TCII production is rate of limiting for cbl absorption. Methods used will include transfection of eukaryotic cells, transformation of prokaryotic cells, deletional and in situ mutagenesis analysis, cell fractionation of enterocytes and tissue culture cells (Core C), immunolocalization and autoradiography (Core B), and CDNA sequencing and oligonucleotide production (Core A).
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