The overall objectives of this application are to define the molecular and cellular basis of receptor-mediated endocytosis and transport of vitamin B12/cobalamin (Cbl). Dietary Cbl is absorbed by ileal enterocytes via a specific intrinsic factor-Cbl receptor (IFCR). Cbl is trancytosed across these cells, enters the circulation and is transported to peripheral tissues bound to transcobalamin II (TCII) whereupon the complex is taken up via TCII receptors (TCIIR). Cbl deficiency may arise at a number of different sites such as reduced or defective synthesis of TCII, IFCR, TCII-R or IF in the gastric mucosa or as the result of defective intracellular sorting. Recent studies in the applicant's laboratory have been aimed at characterizing the intestinal mechanisms of Cbl uptake and intracellular transport. Preliminary data suggests that Cbl can enter the circulation not only via the well-known IFCR pathway but also via apically derived TCIIR.
The specific aims of this proposal will expand on these observations and other preliminary data showing isolation of putative cDNA clones of IFCR and TC IIR by: 1) studying the intracellular pathways of Cbl sorting during IF-mediated apical to basolateral transcytosis; 2) characterizing the intracellular sorting pathways for Cbl and TCII following internalization from apical and basolateral domains; 3) studying the route and kinetics of delivery of IFCR and TC II-receptors to their respective plasma membranes; and 4) studying developmental, regional and hormonal regulation of Cbl receptors and initiating studies of structure-function relationships of Cbl receptors by isolating and expressing cDNA clones encoding IF-Cbl and TC-II receptors. The human intestinal-derived Caco-2 cell line will be used as a model for the majority of studies proposed in aims 1-3 and in regulatory studies proposed in aim 4. Opossum kidney (OK) cells, which appear to bind constant and relatively high levels of radiolabeled cobalamin-IFCR will be used to determine whether or not sorting mechanisms are similar in intestinal and kidney-derived cell lines. In vivo studies of rat intestinal uptake of 125I-labeled TC II-Cbl and of differentially labeled IF-Cbl and TCII-Cbl models will be used in an attempt to confirm that Cbl can indeed be transported bound to TC II without degradation during transit through the GI tract to the distal small intestine. Caco-2 cells were chosen because although ICFR binding is lower and does vary greatly with passage, cobalamin-TCII-R binding and antibody cross-reactivity with a 124 kDa membrane protein on Westerns appears to be approximately 10 times greater than in OK cells. Planned experimental approaches to address the aims of this proposal range from standard biochemical applications such as radiolabeled cobalamin binding assays, protein isolation and purification, antibody generation, immunoprecipitation, density gradient separations, Western and Northern analyses to more sophisticated and technically demanding free flow electrophoresis, receptor cloning, in vitro transcription and translation and Xenopus expression protocols. The revised specific aim 4 also now includes in vitro mutagenesis for identification of ligand binding regions and the cytoplasmic basolateral targeting domain(s) of TC II-R. Effects of mutations on basolateral targeting of TC II-R will be examined by cloning into the expression vector, pSFFV-neo, and transfection into Caco-2 cells.
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|Kalra, Seema; Li, Ning; Yammani, Raghunatha R et al. (2004) Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin. Arch Biochem Biophys 431:189-96|
|Yammani, R R; Seetharam, S; Dahms, N M et al. (2003) Transcobalamin II receptor interacts with megalin in the renal apical brush border membrane. J Membr Biol 193:57-66|
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|Vanamala, Sravan K; Seetharam, Shakuntla; Yammani, Raghunatha R et al. (2003) Human transcobalamin II receptor binds to Staphylococcus aureus protein A: implications as to its structure and function. Arch Biochem Biophys 411:204-14|
|Kalra, Seema; Li, Ning; Seetharam, Shakuntla et al. (2003) Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187. Am J Physiol Cell Physiol 285:C150-60|
|Bauer, Joseph A; Morrison, Bei H; Grane, Ronald W et al. (2002) Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin. J Natl Cancer Inst 94:1010-9|
|Seetharam, B; Li, N (2000) Transcobalamin II and its cell surface receptor. Vitam Horm 59:337-66|
|Seetharam, B (1999) Receptor-mediated endocytosis of cobalamin (vitamin B12). Annu Rev Nutr 19:173-95|
|Seetharam, B; Bose, S; Li, N (1999) Cellular import of cobalamin (Vitamin B-12). J Nutr 129:1761-4|
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