An important function of the liver is transport and metabolism of various molecules from the circulation. Much of this transport activity resides in the hepatocyte. The focus of our research has been elucidation of two distinct, physiologically important hepatocellular transport mechanisms, one for organic anions and the other for desialylated glycoproteins.
Organic anion transport is an important mechanism by which these molecules can be removed from the body. These anions tend to be hydrophobic and circulate bound to serum albumin. They include drugs, steroid hormones, bile acids, and xenobiotics. They are extracted from albumin very quickly by the hepatocyte and enter the cell by transmembrane movement. We have elucidated a number of important characteristics of this transporter in a cultured rat hepatocyte system, using sulfobromophthalein (BSP) as a prototypic substrate. The hallmark of this process is the chloride-dependent extraction of organic anion from albumin. These findings were used to devise a functional assay for organic anion transport which we utilized to clone the transporter in a Xenopus oocyte expression system. Northern blot analysis under high stringency revealed hybridization to RNA extracted only from kidney and liver, although recent studies have also demonstrated RNA in choroid plexus by RT-PCR and in situ hybridization. This protein is the first member of a new family of transporters and has been named organic anion transport protein 1 (oatp1). Computer modeling reveals that the peptide sequence can be best represented by 12 transmembrane domains, with both amino and carboxy termini localized intracellularly. There are 3 potential extracellular glycosylation sites. We have prepared antibodies to oatp1 and have found that, in liver, it is a hepatocyte specific basolateral plasma membrane glycoprotein of 80 kDa. Interestingly, in kidney, it is an 83 kDa apical plasma membrane glycoprotein localized to the S3 segment of the proximal tubule, and migrates as 37 and 35 kDa peptides on SDS-PAGE performed under reducing conditions. There is no effect of reduction on migration of liver oatp. In choroid plexus, oatp migrates as an 80 kDa protein localized to the apical (CSF) choroid surface. The mechanisms for differential processing and targeting of oatp in these cells is now under study. In addition, transport activity of oatp1 is rapidly down-regulated by serine phosphorylation, and requires exchange with an intracellular anion such as bicarbonate. We have prepared stable transfectants of oatp in HeLa and MDCK cells. Future investigation will examine structure-function relationships as well as the topological orientation of the protein in the plasma membrane using appropriate molecular and biochemical tools.
In contrast to the mechanism of organic anion uptake, uptake of ligands such as desialylated glycoproteins is by receptor-mediated endocytosis and involves internalization of ligand-receptor complexes into an endocytic vesicle (endosome). Subsequently, these complexes dissociate as the endosome acidifies, and ligand and receptor segregate into separate compartments. Ultimately ligand traffics to the lysosome where it is degraded, while receptor recycles to the cell surface where it is reutilized. Our previous studies have shown that this segregation process requires integrity of microtubules. We are investigating the role of microtubules in providing a directed path for these processes and the potential importance of microtubule-associated motor molecules such as kinesin and dynein in providing the force for vesicular movement. In recent studies, we have devised a cell free in vitro system to dissect the functional components of these processes. In this system, endocytic vesicles on microtubules can be viewed using microscopic technology that permits quantitation of direction and rates of movement. This technology has now been optimized and has also permitted the reconstitution of vesicle fission and segregation. Future studies are aimed at dissecting the molecular bases of these processes.
Selected References
NCBI PubMed search of "A.W. Wolkoff"
Shi, X., Bai, S., Ford, A.C., Burk, R.D., Jacquemin, E., Hagenbuch, B., Meier, P.J., and Wolkoff, A.W. (1995) Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells. J. Biol. Chem. 270:25591-25595.
Hogue-Angeletti, R., Novikoff, P.M., Juvvadi, S.R., Fritschy, J-M., Meier, P.J., and Wolkoff, A.W. (1997) The choroid plexus epithelium is the site of the organic anion transport protein in the brain. Proc. Natl. Acad. Sci., USA 94:283-286.
Kanai, N., Lu, R., Satriano, J.A., Bao, Y., Wolkoff, A.W. and Schuster, V.L. (1995) Identification and characterization of a prostaglandin transporter. Science 268:866-869.
Novikoff, P.M., Cammer, M., Tao, L., Stockert, R.J., Wolkoff, A.W. and Satir, P. (1996) Three-dimensional organization of rat hepatocyte cytoskeleton: Relation to the asialoglycoprotein endocytosis pathway. J. Cell Sci. 109:21-32.
Oda, H., Stockert, R.J., Collins, C., Wang, H., Novikoff, P.M., Satir, P. and Wolkoff, A.W. (1995) Interaction of the microtubule cytoskeleton with endocytic vesicles and cytoplasmic dynein in rat cultured hepatocytes. J. Biol. Chem. 270:15242-15249.
Satlin, L.M., Amin, V. and Wolkoff, A.W. (1997) Organic anion transporting polypeptide (oatp) mediates organic anion/HCO3- exchange. J. Biol. Chem. 272:26340-26345.
Hogue-Angeletti, R., Bergwerk, A.J., Novikoff, P.M., Wolkoff, A.W. (1998) Dichotomous development of the organic anion transport protein in liver and choroid plexus. Am. J. Physiol., 275:C882-C887.
Glavy, J.S., Wu, S.M., Wang, P.J., Orr, G.A., and Wolkoff, A.W. (2000) Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1. J. Biol. Chem. 275:1479-1484.
Murray, J.W., Bananis, E., and Wolkoff, A.W. (2000) Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bi-directional process. Mol. Biol. Cell 11:419-433.
Bananis, E., Murray, J.W., Stockert, R.J., Satir, P., and Wolkoff, AW. (2000) Microtubule and motor-dependent endocytic vesicle sorting in vitro. J Cell Biol. 151:179-86.
