Expression Of Plasma Membrane Proteins Research Articles

P9 Altered expression of plasma membrane proteins on breast cancer cells capable of forming metastasis. Identification by comparative proteomic analysis

P9 Altered expression of plasma membrane proteins on breast cancer cells capable of forming metastasis. Identification by comparative proteomic analysis

Identification of exocytosis mediator proteins in peripheral blood neutrophils of patients with chronic myeloid leukemia.

We demonstrated expression of plasma membrane proteins (syntaxin-4 and syntaxin-6) and specific/gelatinase granule membrane proteins (SNAP-25 and VAMP-2) in the peripheral blood neutrophils of patients with chronic myeloid leukemia. VAMP-1 associated with membranes of azurophilic and specific/gelatinase granules was absent in peripheral blood neutrophils of patients with chronic myeloid leukemia. Decreased capacity of neutrophils to exocytosis in chronic myeloid leukemia is probably caused by the absence of VAMP-1 in these cells.

The maintenance and generation of membrane polarity in hepatocytes.

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal– basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking.We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1,2

Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma × fibroblast hybrids and in hepatoma dedifferentiated variants

We have studied two aspects of the plasma membrane of hepatocytes, highly differentiated epithelial cells that exhibit a particular and complex polarity. Using a genetic approach, we have distinguished between the expression/regulation of proteins specific for all three hepatocyte membrane domains and their organization into discrete domains. For this analysis we used a panel of previously isolated cell clones, derived from the differentiated rat hepatoma line H4IIEC3, and that present different expression patterns for liver-specific genes. This panel was composed of (1) differentiated clones, (2) chromosomally reduced hepatoma-fibroblast hybrids characterized by a pleiotropic extinction/reexpression of liver-specific genes and (3) dedifferentiated variant and revertant clones. The expression of 16 hepatocyte membrane polarity markers was studied by western blotting and immunolocalization. Even though cells of differentiated clones express all of these polarity markers, they are not polarized, and are therefore suitable for studying the regulation of plasma membrane protein expression, and for identifying gene products implicated in the establishment of membrane polarity. In hepatoma-fibroblast hybrids the expression of four markers, three apical (dipeptidylpeptidase IV, alkaline phosphodiesterase B10 and polymeric IgA receptor) and one lateral (E-cadherin), is down-regulated in extinguished clones and restored in reexpressing subclones, as previously reported for liver-specific functions. The dipeptidylpeptidase IV mRNA was undetectable or strongly reduced in extinguished hybrids, but expressed at a robust level in some of the reexpressing clones. Concerning the dedifferentiated variants, each has its own pattern of membrane marker expression (loss of expression of three to six markers), that differs from that of extinguished hybrids. Revertant cells express all of the membrane markers examined. Among all of these hepatoma derivatives, only cells of reexpressing hybrids are polarized, and form bile canaliculi-like structures, with spherical and even, for one clone, long tubular and branched forms. All apical markers examined are confined in these canalicular structures, whereas the other markers are excluded from them, and present on the rest of the membrane (basolateral markers) or at the cell-cell contacts (lateral markers). Cells of reexpressing hybrids also express simple epithelial polarity. Thus the expression of only a few hepatocyte-domain-specific plasma membrane proteins is subject to down-regulation, as is the case for liver-specific genes so far studied, and the expression of polarity markers and the formation of poles are dissociable events.

Effects of Vpu Expression onXenopusOocyte Membrane Conductance

The HIV-1-specificvpugene encodes an integral membrane phosphoprotein which affects three aspects of the HIV-1 infectious cycle: it enhances virion release from infected cells; it causes degradation of the CD4 protein in the endoplasmic reticulum; and it delays syncytia formation in HIV-1-infected CD4+T-cells. Although little is known about how Vpu mediates these effects, it has been proposed to function as a nonspecific cation channel. In this report, voltage clamp measurements ofXenopusoocytes show that Vpu expression is not associated with increased transmembrane currents. Instead, Vpu expression diminishes membrane conductance. Injection of 4.6 ng of Vpu mRNA into these cells reduces endogenous potassium conductance by 50%. Only Vpu mutants which retain the ability to degrade CD4 can diminish K+conductance. Inhibition by Vpu is not unique to K+channels as it is also observed on several coexpressed membrane proteins but not on a coexpressed cytoplasmic protein. These results indicate that the CD4 degradative capability of Vpu and the Vpu-mediated modulation of membrane protein expression are mechanistically coupled and that Vpu may contribute to HIV pathogenesis by altering plasma membrane protein expression at the cell surface.

The plasma membrane polarity of human biliary epithelial cells: in situ immunohistochemical analysis and functional implications

Background/Aims: In transporting epithelia, like the biliary epithelium, most plasma membrane proteins present a polarized distribution, essential for the maintenance of the structural and functional properties of the epithelium. We therefore analyzed the expression of polarized plasma membrane proteins by human biliary epithelial cells in order to compare them with other transporting epithelial cells and to search for differences in plasma membrane protein expression between their different anatomical subsets. Methods: We designed an in situ immunohistochemical study of the various anatomical compartments of the human biliary tract in order to assess the pattern of expression of selected polarized plasma membrane proteins, including integrin receptors, ectopeptidases, membrane transporters and GPI-linked proteins. Results: All biliary epithelial cells expressed the same repertoire of integrins, except for integrin chain α5, restricted to the intra-hepatic compartments. All biliary epithelial cells expressed the following apical ectopeptidases: aminopeptidase-N, neutral-endopeptidase, dipeptidyl-peptidase IV. All biliary epithelial cells epxressed the mebrane transporter Na +K +-ATPase, restricted to the basolateral domain, and the apical transporters CFTR and MDR-1. The apical AE2 anion exchanger was restricted to the small intra-hepatic bile ducts and the gallbladder. The GPI-linked protein protectin was basolateral in the intrahepatic bile ducts and apical in the gallbladder. Conclusions: The structural organization of the plasma membrane of biliary epithelial cells is very similar to that of other simple epithelia and exhibits a limited degree of heterogeneity.

Effect of obstructive cholestasis on membrane traffic and domain-specific expression of plasma membrane proteins in rat liver parenchymal cells.

We investigated the effect of bile duct ligation and its release on membrane traffic and plasma membrane protein distribution in rat hepatocytes. Immunofluorescence studies with monoclonal antibodies against six domain-specific surface antigens revealed that bile duct ligation leads to an accumulation of pericanalicular vesicles containing canalicular antigens. All apical antigens could be demonstrated in the basolateral plasma membrane, whereas only one out of three basolateral antigens redistributed to the canalicular plasma membrane. After release of bile duct ligation, the accumulated pericanalicular vesicles disappeared within minutes, whereas the plasma membrane polarity was not restored within 1 hr. Monitoring secretion of polymeric IgA and horseradish peroxidase into bile demonstrated that bile duct ligation also inhibits the transcytotic vesicle pathway and severely impairs the function of tight junctions. In contrast, bile duct ligation appears not to affect the endoplasmic reticulum to basolateral membrane traffic as assessed by determination of newly synthesized albumin and transferrin in serum nor does it influence receptor mediated endocytosis at the basolateral plasma membrane.

Effect of Obstructive Cholestasis on Membrane Traffic and Domain–Specific Expression of Plasma Membrane Proteins in Rat Liver Parenchmal Cells

ABSTRACT We investigated the effect of bile duct ligation and its release on membrane traffic and plasma membrane protein distribution in rat hepatocytes. Immunofluorescence studies with monoclonal antibodies against six domain–specific surface antigens revealed that bile duct ligation leads to an accumulation of pericanalicular vesicles containing canalicular antigens. All apical antigens could be demonstrated in the basolateral plasma membrane, whereas only one out of three basolateral antigens redistributed to the canalicular plasma membrane. After release of bile duct ligation, the accumulated pericanalicular vesicles disappeared within minutes, whereas the plasma membrane polarity was not restored within 1 hr. Monitoring secretion of polymeric IgA and horseradish peroxidase into bile demonstrated that bile duct ligation also inhibits the transcytotic vesicle pathway and severely impairs the function of tight junctions. In contrast, bile duct ligation appears not to affect the endoplasmic reticulum to basolateral membrane traffic as assessed by determination of newly synthesized albumin and transferrin in serum nor does it influence receptor mediated endocytosis at the basolateral plasma membrane. (Hepatology 1994;20:201-212.)

Flow cytometric analysis of African swine fever virus-induced plasma membrane proteins and their humoral immune response in infected pigs

African swine fever (ASF) virus-induced plasma membrane proteins may contribute to the protective immune response against the disease since they can be involved in the antibody-mediated lysis of infected cells. In this study we describe the regulation of ASF virus-induced plasma membrane protein expression and its antibody induction in pigs after viral infection by flow cytometric analysis. More than 80% of infected cells contained viral antigens on the surface membranes at 6 hr postinfection (hpi), and the relative amount of viral antigen expression was increased at 12 and 20 hpi. The kinetics of individual viral protein expression on cell surfaces was studied by a collection of monospecific antibodies directed against the six viral plasma membrane proteins p12, p15, p16, p23.5, p30, and p35. Most of these proteins were expressed at 6 hpi, with the exception of p35, which was first detected at 12 hpi. The percentage of cells expressing each antigen at different hpi was also determined. The immune response against virus-induced plasma membrane proteins in pigs infected with an attenuated ASF virus strain was studied. Antibodies against viral epitopes exposed on plasma membranes reached a plateau at 20 days postinfection (dpi). The relative amount of antibodies induced during infection with these specificities was not directly related to the antibody titer of the sera. Sera obtained at 20 and 40 dpi contained antibodies against most of the viral plasma membrane proteins and were most efficient in recognition of viral antigens exposed on the surface of infected cells at early times.

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Use ofXenopus oocytes for the functional expression of plasma membrane proteins

Use ofXenopus oocytes for the functional expression of plasma membrane proteins

HORMONAL REGULATION OF LIVER PLASMA MEMBRANE PROTEIN EXPRESSION DURING DEVELOPMENT

In the past, dexamethasone and glucagon has been shown to have a variable effect on the expression of soluble liver enzymes during development. The aim of the present study was to investigate the effect of dexamethasone and glucagon on the expression of plasma membrane proteins during ontogeny. Our previous studies have demonstrated a significant increase of the asialoglycoprotein receptor (ASGR) and 110,000 Mr glycoprotein at birth, decrease in alkaline phosphatase (AP) and gammaglutamyltranspeptidase (GT) and very little change in leucine aminopeptidase (LAP) after birth. Buffalo rat fetuses at 18 days of gestation and 1-day-old newborns were injected (single and daily, respectively) with dexamethasone (2 and 4 μg, respectively) or glucagon (25 and 50 μg, respectively). Fetuses were sacrificed at 22 days of gestation and newborns at 3, 5, and 7 days of age. Total post-nuclei membrane vesicles were prepared from rat livers using a sucrose gradient method. Quantitation of levels of membrane proteins was performed using immunological and/or enzymatic methods. In fetal rats, dexamethasone did not affect the expression of membrane proteins. In contrast, glucagon increased significantly the levels of AP, LAP, and GT. In newborns, glucagon did not alter membrane protein levels. However, dexamethasone dramatically increased six-fold the level of GT while the amounts of ASGR, AP, and LAP were reduced by two-to four-fold. These data indicate that there is a differential effect of dexamethasone and glucagon on the expression of membrane proteins during late intrauterine and early extrauterine stages of development.