Hepatocytes in the mammalian liver are polarized epithelial cells having a plasma membrane with apical, lateral, and basal domains separated by tight junctions. The basal and lateral domains of the hepatocyte are usually grouped together as the basolateral or sinusoidal domain while the bile canalicular domain of the hepatocyte plasma membrane is considered analogous to the apical domain of other polarized cells. The receptor system that recognizes galactose-terminated glycoproteins, or asialo-glycoproteins, is a major constituent of the sinusoidal plasma membrane domain of hepatocytes. Dipeptidyl peptidase IV and a protein of Mr 110,000 with ecto-ATPase activity are major constituents of the bile canalicular plasma membrane domain of hepatocytes. The overall goal of the proposed research is to understand the cellular mechanisms used for the biogenesis, delivery to, and maintenance of these different domain-specific glycoproteins in their correct domain of residence in the plasma membrane of the hepatocyte. A variety of methods including cell fractionation, immunological, gene transfection, and mutagenesis are being used to accomplish the overall goal. Full-length cDNAs for each of the domain-specific glycoproteins will be transfected into either stable polarized cell lines in culture or into primary cultures of mouse hepatocytes. The pathway of biogenesis of the transfected gene products will be analyzed and compared to the pathway of biogenesis of the same protein in primary cultures of rat hepatocytes or to the equivalent endogenous proteins in the stable cell lines in culture. Mutations resulting from deletions and site-directed mutagenesis will be used to identify determinants in either the amino acid sequences or structures of the different domain-specific proteins that are responsible for correct localization in the plasma membrane of the transfected polarized cell.
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