Surface Biotinylation Studies Research Articles

Altered subcellular distribution of Na+,K+-ATPase in proximal tubules in young spontaneously hypertensive rats.

During early development of hypertension, the spontaneously hypertensive rat (SHR) demonstrates increased proximal tubule sodium reabsorption. Our previous observations of reduced Na+,K+-ATPase catalytic alpha1 and gamma subunit transcript abundance in SHR proximal tubule led us to test the hypothesis that increased proximal tubule sodium reabsorption may be attributable to altered subunit protein abundance, post-translational modification, or a shift in subcellular alpha1 and gamma distribution toward the basolateral membrane. We now extend previous gene expression studies by analyzing total cellular alpha1 and gamma protein abundance in proximal tubule from SHR compared with matched Wistar-Kyoto (WKY) controls. We also used sucrose density-gradient centrifugation to isolate basolateral, early, and late endosomal membrane-enriched fractions as well as cell surface biotinylation to test the hypothesis of altered subunit subcellular distribution in the SHR proximal tubule. At 4 weeks of age, significantly greater amounts of alpha1 were present in basolateral membrane-enriched fractions of SHR than WKY (21.1+/-1.8% versus 12.3+/-1.8%; P<0.005), and there was a concomitant reduction of alpha1 in late endosomal membrane-enriched fractions of SHR (63.3+/-2.7% versus 74.8+/-4.3%; P<0.05). This finding was confirmed in cell surface biotinylation studies that showed higher alpha1 (1.45+/-0.1-fold greater; P<0.05) and gamma-subunit (3.48+/-0.7-fold greater; P<0.01) abundance in 4-week-old SHR proximal tubule plasma membrane compared with matched WKY samples. These studies support the hypothesis that development of hypertension in SHR may involve an altered subcellular distribution of proximal tubule Na+,K+-ATPase subunits.

Expression of the zinc transporter ZnT4 is decreased in the progression from early prostate disease to invasive prostate cancer.

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.

N-glycosylation is required for the surface localization of MUC17 mucin

The nucleic acid sequence of the human gene, MUC17, indicates that this mucin contains an SEA domain, a transmembrane domain, and putative N-glycosylation sites in the carboxyl terminus. Mucins that possess an SEA domain are usually proteolytically cleaved within that domain to yield two subunits, the smaller of which is associated with the surface membrane. Homogenates of ASPC-1 pancreatic cancer cells showed three main bands of immunoreactivity with alpha-SEA (a polyclonal antibody directed against a site downstream of the postulated cleavage site) after SDS-PAGE and Western blotting (38, 45, and 49 kDa). Experiments utilizing N-glycan specific hydrolases showed that the 38 kDa band contained high mannose glycans whereas the 45 and 49 kDa bands contained complex-type glycans. Only two smaller alpha-SEA reactive bands (30 and 32 kDa) were present after cells had been treated with the N-glycosylation inhibitor tunicamycin. Surface biotinylation studies showed that only the forms possessing complex-type N-glycans were localized to the cell surface. Both tunicamycin and brefeldin A, an inhibitor of protein transport, reduced surface localization. In summary, our results indicate that the surface localization of the smaller subunit of MUC17 is dependent on its N-glycosylation status.

A human brain tumor-derived PDGFR-alpha deletion mutant is transforming.

Aberrant receptor tyrosine kinase signaling plays an important role in the molecular pathogenesis of brain tumors. We have been studying a previously identified human glioblastoma-derived PDGFR-alpha mutant that has an in-frame deletion in the extracellular domain, causing loss of exons 8 and 9 (PDGFR-alpha(delta8,9)). In the primary tumor, this deletion mutant receptor was shown to be amplified and overexpressed. The purpose of this study was to determine the expression, activity, localization, and transformation properties of this deletion mutant. In the absence of serum, or PDGF-AA, PDGFR-alpha(delta8,9) was phosphorylated on tyrosine residues, indicating ligand-independent autoactivation. Localization by staining and cell surface biotinylation studies revealed expression of the deletion mutant predominantly in the cytoplasm, with very little present on the cell surface. To determine if PDGFR-alpha(delta8,9) was oncogenic, we transfected wild-type and mutant receptors into Rat1 cells and performed analyses of cell growth, in vitro transformation, and subcutaneous growth in the nude mouse. PDGFR-alpha(delta8,9)-expressing cells displayed enhanced cell growth and survival in low serum, and formed foci in monolayer cultures. PDGFR-alpha(delta8,9)-expressing Rat1 cells were also tumorigenic when injected subcutaneously into nude mice. Expression of PDGFR-alpha(delta8,9) was also associated with increased c-Jun phosphorylation in the absence of PDGF ligand, demonstrating also that the mutant receptor is associated with altered intracellular signaling. These data demonstrate that PDGFR-alpha(delta8,9) is transforming, and it is the first demonstration of a naturally occurring tumor-derived mutant PDGFR-alpha with oncogenic properties.

ABC transporters in liver, kidney, and intestine

ABC transporters in liver, kidney, and intestine

Na(+)/H(+) exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells.

Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle.

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 +/- 9.4 microM and maximal velocity value 17.3 +/- 3.1 pmol x min(-1) x mg vesicle protein(-1)), which cotransports Cr into skeletal muscle together with Na(+) and Cl(-) ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH(2)-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of approximately 58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [(14)C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells.

Recombinant protein produced from a cDNA cloned in our laboratory (UAT) functions in lipid bilayers as a urate transporter/channel. Because UAT is a galectin, a family of proteins presumed to be soluble, the localization and topology of UAT were assessed in living cells. UAT was targeted to plasma membrane in multiple epithelium-derived cell lines and, in polarized cells, was targeted to both apical and basolateral membranes. The amino and carboxy termini of UAT were both detected on the cytoplasmic side of plasma membranes, whereas cell surface biotinylation studies demonstrated that UAT is not merely a cytosolic membrane-associated protein but contains at least one extracellular domain. Madin-Darby canine kidney cells were shown both functionally and immunologically to contain an apparent homolog of UAT; however, transfection with UAT did not modify urate uptake. Because coimmunoprecipitation studies revealed that UAT is capable of forming both homo- and heteromultimers, it is proposed that monomers of endogenous channels are in part replaced by monomers of the protein expressed subsequent to transfection, thereby maintaining constancy of urate uptake at basal levels.

STIM1: a novel phosphoprotein located at the cell surface

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

The short forms of HLA-G are unlikely to play a role in pregnancy because they are not expressed at the cell surface

HLA-G is a nonclassical class I MHC molecule of unknown function expressed on human invasive trophoblast. In trophoblast cells, HLA-G mRNA is alternatively spliced into a variety of forms which are predicted to encode a full length membrane-bound form, three short membrane-bound isoforms and two soluble isoforms. The aim of this study was to determine which of these protein isoforms are translated, which are expressed on the cell surface and which are secreted. Artificial cDNAs encoding the isoforms were generated by PCR mutagenesis, ligated to an epitope tag and transfected into a human cell line capable of expressing MHC class I. Protein products of appropriate sizes were detected in cells transfected with cDNAs encoding all membrane-bound forms, but surface biotinylation studies indicated that only full length membrane-bound HLA-G was present at the cell surface. Full length HLA-G was also detected by surface antibody binding and flow cytometry. Soluble HLA-G1 was detected in cells transfected with the appropriate cDNA only after treatment with monensin, which inhibits transport of glycoproteins through the Golgi apparatus. These results suggest that full length HLA-G, but not short HLA-G isoforms can be expressed on the surface of human cells and that soluble HLA-G is rapidly secreted. Thus, it is likely that the full length membrane-bound and soluble forms of HLA-G are the only biologically active forms to which the mother is exposed.

Intracellular formation and processing of the heterotrimeric gH-gL-gO (gCIII) glycoprotein envelope complex of human cytomegalovirus.

The human cytomegalovirus (HCMV) gCIII complex contains glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and glycoprotein O (gO; gpUL74). To examine how gH, gL, and gO interact within HCMV-infected cells to assemble the tripartite complex, pulse-chase experiments were performed. These analyses demonstrated that gH and gL associate by the end of the pulse period to form a disulfide dependent gH-gL complex. Subsequently, the gH-gL complex interacts with a 100-kDa precursor form of gO to form a 220-kDa precursor of the mature gH-gL-gO complex that contains a 125-kDa form of gO. The 220-kDa precursor complex (pgCIII) was sensitive to treatment with endoglycosidase H (endo H), while the mature gCIII complex was essentially resistant to digestion with this enzyme, suggesting that formation of pgCIII complex occurs in the endoplasmic reticulum (ER) and is processed to mature gH-gL-gO (gCIII) in a post-ER compartment. While the N-linked glycans on the 100-kDa form of gO were modified to endo H-resistant states as the 125-kDa gO formed, additional posttranslational modifications were detected on gO. These processing alterations were non-N-linked oligosaccharide modifications that could not be accounted for by phosphorylation or by O-glycosylation of the type sensitive to O-glycanase. Of gH, gL, gO, and the various complexes that they form, only the mature form of the complex was detectable at the infected cell membrane, as judged by surface biotinylation studies.

Cell Surface Ectodomain Cleavage of Human Amphiregulin Precursor Is Sensitive to a Metalloprotease Inhibitor: RELEASE OF A PREDOMINANT N-GLYCOSYLATED 43-kDa SOLUBLE FORM

Biosynthesis and processing of amphiregulin (AR) have been investigated in human colorectal (HCA-7, Caco-2) and mammary (MCF-7) cancer cell lines, as well as in Madin-Darby canine kidney cells stably expressing various human AR precursor (pro-AR) forms. Both cells expressing endogenous and transfected AR produce multiple cellular and soluble forms of AR with an N-glycosylated 50-kDa pro-AR form being predominant. Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominant N-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length N-terminal pro-region, which may influence the biological activities of these forms.

Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.

We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.e., the time of plating) or Day 4 or 6 through Day 8 in culture. AECs monolayers express AQP5 only on their apical surfaces as determined by cell surface biotinylation studies. Control AECs grown in the absence of KGF through Day 8 express increasing levels of AQP5, consistent with transition toward the AT1 cell phenotype. Exposure of AECs to KGF from Day 0 results in decreased AQP5 expression, retention of a cuboidal morphology, and greater numbers of lamellar bodies relative to control on Day 8 in culture. AECs treated with KGF from Day 4 or 6 exhibit a decrease in AQP5 expression through subsequent days in culture, as well as an increase in expression of surfactant apoproteins. These data, showing that KGF both prevents and reverses the increase in AQP5 (and decrease in surfactant apoprotein) expression that accompanies progression of the AT2 toward the AT1 cell phenotype, support the concepts that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible and that KGF may play a major role in modulating AEC phenotype.

Na:K:2Cl Cotransporter (NKCC) of Intestinal Epithelial Cells: SURFACE EXPRESSION IN RESPONSE TO cAMP

During intestinal chloride secretion, epithelial uptake of salts is accomplished largely by a bumetanide-sensitive Na:K:2Cl cotransporter designated here as NKCC. Using monoclonal antibodies directed against NKCC from the human crypt epithelial cell line, T84, we define its surface localization as a function of cotransporter activation. Immunoelectron microscopy, confocal localization, and selective surface biotinylation studies revealed that the 195-kDa NKCC protein is polarized to the basolateral domain. Following immunoprecipitation, several polypeptides coprecipitated with the 195-kDa cotransporter including two prominent proteins of molecular mass 160 and 130 kDa. Immunoblotting with three distinct anti-NKCC monoclonal antibodies in conjunction with deglycosylation experiments suggested that the 160- and 130-kDa bands represented novel proteins unrelated to the cotransporter. Stimulation of T84 monolayers with cAMP agonists, a condition which elicits chloride secretion and leads to microfilament-dependent NKCC activation, did not significantly increase the number of bumetanide-binding sites and only marginally increased surface expression of the 195-kDa cotransporter available for surface biotinylation. In contrast, cAMP agonist stimulation increased the surface expression of the coprecipitating 160- and 130-kDa proteins approximately 6-fold. The increase in surface 160- and 130-kDa proteins was attenuated by phalloidin preloading the cells, a condition which also prevents activation of NKCC without influencing the activity of other membrane transporters participating in chloride secretion. These studies define the polarized distribution of the NKCC protein on intestinal epithelia, indicate that NKCC may be associated with two other previously unidentified membrane proteins and such association is influenced by the F-actin cytoskeleton.