The overall objective of this proposal is to define the mechanisms involved in the intracellular movement of cobalamin (cbl) following its cellular uptake, by examining the biology of the two major receptors for cbl binding proteins. We will use existing intrinsic factor (IF)-cbl receptor antiserum and raise antiserum to Transcobalamin II (TC II) receptor. These studies will be carried out using tissue explants or polarized kidney (MDCK) and intestine (Caco-2) cells. Intracellular transit, distribution and carbohydrate processing will be assessed by pulse chase experiments using 3H amino acids and sugars. The trafficking of the receptor will be monitored on SDS PAGE using immunoprecipitates of the labeled receptor extracted from various organelles during its transit in the cell. We will determine whether the receptor undergoes specialized post translational modification such as fatty acid acylation. If receptor is indeed acylated we will characterize the domains which are acylated and the nature (ester vs amide) of the linkage with the fatty acid. The nature of lipid-protein interaction important for anchoring, and activity of the receptor will be examined using artificial bilayer systems and the pure receptor. The nature and site of membrane insertion (NH2 or COOH terminus) will be examined. In order to probe further into the structure of the IF-cbl receptor and its possible homology with the ligand (IF), we will sequence selected fragments of the receptor obtained by proteolysis, and prepare oligonucleotide probes. We will screen a size fractionated lambda gtll cDNA rat kidney library with antibody and oligonucleotide probes, in order to isolate a cDNA clone encoding the IF-cbl receptor. The clone will be used to elucidate receptor structure by DNA sequencing and biosynthesis, by dot blot analysis of tissues and cell lines. Using culture normal and transformed cells, we will study the uptake and movement of free and protein bound cbl in order to study a) intracellular fate of IF, b) formation of TC II-cbl, c) the role of TC II-receptor in the exocytosis of cbl. These studies are designed to understand the intracellular events which regulate the synthesis of proteins involved in cbl uptake, and could provide clues in understanding the mechanism of cbl deficiency noted in patients with familial cbl malabsorption and defects in the intracellular routing of cbl.
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