We have identified a 48-kD basolateral-specific integral plasma membrane sialoglycoprotein of rat hepatocytes -- called CE 9 -- whose expression and modification are regulated by the group of hypolipidemic drugs and phthalate-ester plasticizers known collectively as the peroxisome proliferators (PPs). Even short-term dietary exposure of rats to PPs induces the expression of a new isoform of CE 9 in hepatocytes while simultaneously causing the down-regulation of many of the other domain- specific integral plasma membrane proteins of the hepatocyte. The PP- induced isoform of CE 9 exhibits a lower molecular mass and a more basic isoelectric point which reflect in part an altered pattern of glycosylation and a decrease in the level of phosphorylation. We are actively investigating the molecular basis for these plasma membrane-directed aspects of the hepatocyte's pleiotropic response to this group of medically and environmentally important hepatocarcinogens. We are pursuing the hypothesis that CE 9 is a plasma membrane transport or binding protein for the PPs. To aid us in our efforts, we have recently isolated a full-length CE 9 cDNA from a rat liver lambda gt11 library. We propose to: (1) complete the sequencing of our full-length rat liver CE 9 cDNA and construct a molecular map of CE 9 which highlights its extracellular, transmembrane and cytoplasmic domains, potential sites of glycosylation or phosphorylation, and any other instances of amino acid sequence similarity which might help to clarify the function of CE 9 and its modes of regulation by the PPs; (2) use of cDNA probes to determine whether the PP- induced increases in the concentration and in the rate of biosynthesis of CE 9 protein in rat liver reflect changes in transcription or mRNA accumulation; (3) use cDNA and antibody probes to determine whether the multiple isoforms of CE 9 observed in different rat tissues are the products of different genes and/or mRNAs or whether they arise through differential co- and/or posttranslational processing; (4) use immunoprecipitation, [32P] phosphopeptide mapping and [32P]phosphoamino acid analysis to identify the peptide sites, amino acids and protein kinases involved in the phosphorylation of CE 9 and its regulation by PPs; and (5) use affinity techniques to identify instances of direct interaction between CE 9 and the PPs and use anti-CE 9 antibody to examine the role of CE 9 in the binding and uptake of radiolabeled PP in the isolated perfused rat liver.
| Fayos, B E; Bartles, J R (1994) Regulation of hepatocytic glycoprotein sialylation and sialyltransferases by peroxisome proliferators. J Biol Chem 269:2151-7 |
| Nehme, C L; Cesario, M M; Myles, D G et al. (1993) Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon. J Cell Biol 120:687-94 |
