Membrane-associated Form Research Articles

Expression of membrane-associated mucins MUC1 and MUC4 in major human salivary glands.

Mucins are high molecular weight glycoproteins secreted by salivary glands and epithelial cells lining the digestive, respiratory, and reproductive tracts. These glycoproteins, encoded in at least 13 distinct human genes, can be subdivided into gel-forming and membrane-associated forms. The gel-forming mucin MUC5B is secreted by mucous acinar cells in major and minor salivary glands, but little is known about the expression pattern of membrane-associated mucins. In this study, RT-PCR and Northern blotting demonstrated the presence of transcripts for MUC1 and MUC4 in both parotid and submandibular glands, and in situ hybridization localized these transcripts to epithelial cells lining striated and excretory ducts and in some serous acinar cells. The same cellular distribution was observed by immunohistochemistry. Soluble forms of both mucins were detected in parotid secretion after immunoprecipitation with mucin-specific antibodies. These studies have shown that membrane-associated mucins are produced in both parotid and submandibular glands and that they are expressed in different cell types than gel-forming mucins. Although the function of these mucins in the oral cavity remains to be elucidated, it is possible that they both contribute to the epithelial protective mucin layer and act as receptors initiating one or more intracellular signal transduction pathways.

No soluble common cytokine receptor gamma chain (γ c) in activated human lymphocyte cultures—comparison with soluble IL-2Rα

The presence of a soluble form of the common cytokine receptor γ chain (γ c) in cell free supernatants from unstimulated and 1–6 days PHA stimulated peripheral blood lymphocyte (PBL) cultures was analyzed using a sandwich ELISA. No naturally produced soluble γ c could be detected in these cell free culture supernatants, although a sensitivity in the nanogram range was achieved for recombinant baculovirus expressed human soluble γ c with this assay (detection limit 0.2 ng hIL-2 sRγ). Analysis of the very same supernatants for soluble IL-2Rα demonstrated increased concentrations (up to 20.4 ng/ml) of this other IL-2R member. The membrane-associated form of the common cytokine receptor γ chain was detected in cell lysates prepared from stimulated PBL at a concentration of 3.5 ng per 0.5×10 6 cells. Analysis of a small panel of serum samples from patients with different disorders verified that the soluble form of hIL-2Rα, but not hIL-2 sRγ, can be detected, which thereby strongly suggests that the human soluble γ c seems to be a valuable marker only for a limited number of clinical disorders.

Role of carbon dioxide and ion transport in the formation of sub-embryonic fluid by the blastoderm of the Japanese quail

1. The explanted blastoderm of the Japanese quail was used to explore the role of ions and carbon dioxide in determining the rate of sub-embryonic fluid (SEF) production between 54 and 72 h of incubation. 2. Amiloride, an inhibitor of Na+/H+ exchange, at concentrations of 10-3 to 10-6 M substantially decreased the rate of SEF production when added to the albumen culture medium. N-ethylmaleimide, an inhibitor of V type H+ ATPase, also decreased this rate but only to a small extent at the highest dose applied, 10-3 M. Both inhibitors had no effect on SEF production when added to the SEF. 3. The inhibitors of cellular bicarbonate and chloride exchange, 4-acetamido-4-'isothiocyano-2, 2-'disulphonic acid (SITS) and 4,4'diisothiocyanostilbene-2,2-'disulphonic acid (DIDS), had no effect upon SEF production. 4. Ouabain, an inhibitor of Na+/K+ ATPase, decreased SEF production substantially at all concentrations added to the SEF (10-3 to 10-6 M). Three sulphonamide inhibitors of carbonic anhydrase, acetazolamide, ethoxzolamide and benzolamide, decreased SEF production when added to the SEF at concentrations of 10-3 to 10-6 M. Benzolamide was by far the most potent. Neither ouabain nor the sulphonamides altered SEF production when added to the albumen culture medium. 5. Using a cobalt precipitation method, carbonic anhydrase activity was localised to the endodermal cells of the area vasculosa. The carbonic anhydrase activity was primarily associated with the lateral plasma membranes, which together with the potent inhibitory effect of benzolamide, suggests the carbonic anhydrase of these cells is the membrane-associated form, CA IV. 6. The changes in SEF composition produced by inhibitors were consistent with the production of SEF by local osmotic gradients. 7. It is concluded that a Na+/K+ ATPase is located on the basolateral membranes of the endodermal cells of the area vasculosa, and that a sodium ion/hydrogen ion exchanger is located on their apical surfaces. Protons for this exchanger would be provided by the hydration of CO2 catalysed by the membrane-associated carbonic anhydrase. Furthermore, it is proposed that the prime function of the endodermal cells of the area vasculosa is the production of SEF.

A membrane-associated form of L-DOPA decarboxylase in the white prepuparium and adult eclosion developmental stages of the Mediterranean fruit fly, Ceratitis capitata

L-DOPA decarboxylase (DDC: EC 4.1.1.28) has been extensively studied in both mammals and insects. DDC is generally considered to be a cytosolic enzyme. This work, however, presents evidence on the association of DDC molecules with the membranes of the white prepuparium and adult eclosion developmental stages of Ceratitis capitata (Mediterranean fruit fly). Membrane-associated DDC immunoreactivity and enzymatic activity was detected by immunoblotting using polyclonal antisera and enzymatic assay experiments. Fractionation of membranes by temperature-induced phase separation in Triton X-114 resulted in the recovery of membrane-associated DDC activity in separation phases where integral and hydrophobic membrane proteins separate. Treatment of membranes with phosphatidylinositol-specific phospholipase C, was not sufficient to remove membrane-associated DDC activity, further suggesting that a population of DDC molecules exists embedded within membranes. In light of the recent evidence describing DDC as a regulated enzyme, a study of the functional significance of the enzyme's association with membranes may provide us with new clues about the biological pathways that this molecule is involved in.

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface.

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

Structurally distinct membrane-associated and soluble forms of GH-binding protein in the mouse.

It has previously been shown that the large increase in GH-binding capacity of mouse liver microsomes during pregnancy is due largely to an increase in the amount of GH-binding protein (GHBP), with a more modest increase in GH receptor (GHR). Here we show that mouse liver GHBP is predominantly present as a membrane-associated protein structurally distinct from the soluble form of GHBP present in serum. Liver GHBP is associated with both intracellular membranes and the plasma membrane. Membrane-associated GHBP and soluble GHBP appear to be identical polypeptides distinguished by the addition of different N-glycans to asparagine residues. The pattern of release of GHBP from membranes by various treatments indicates that GHBP associates with membranes through noncovalent interactions with one or more membrane protein, but not with GHR. Covalent crosslinking provides evidence for several GHBP-associated membrane polypeptides, with molecular masses ranging from 58 kDa to over 200 kDa. These studies in the mouse and similar studies in the rat suggest that GHBP is an important cell-surface receptor for GH in the liver of these species. We postulate that an arginine-glycine-aspartic acid sequence found on rat and mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.

Cre recombinase-controlled expression of the mb-1 allele.

The mb-1 gene is located on chromosome 7 of the mouse genome and it is composed of five exons encoding Ig, which is a B cell-specific protein and an element of the B cell antigen receptor complex (BCR) (Sakaguchi et al., 1998; Hombach et al., 1990). Expression of Igis required for the transport of BCR onto the cell surface. Together with Ig, Igforms a heterodimer that mediates signal transduction from the BCR (Kim et al., 1993). The mb-1 gene is expressed at every stage of B cell development and activation, excluding terminally differentiated plasma cells. The knock-out of mb-1 results in complete block of B cell development at the progenitor stage in mice, as well as in humans (Minegishi et al., 1999; Pelanda et al. unpublished observations). Thus, we created conditional alleles of mb-1 to study the behavior of mature B cells with or without Igexpression. Cre-mediated recombination of inverted loxP sites results in the inversion of the loxP-flanked (floxed) DNA sequence (Nagy 2000). By flanking the mb-1 coding sequence with inverted loxP sites we generated a conditional mb-1 allele that allows switching “on” or “off” (and vice versa) of mb-1 expression under the control of Cre recombinase. In addition, BAP31N-EGFP, which encodes a membrane-associated form of the enhanced green fluorescent protein (mEGFP), was cloned in the opposite transcriptional orientation relative to mb-1 coding sequence into the conditional mb-1 allele. This allows the detection of cells at the “off” state of mb-1 transcription by the expression of mEGFP. Embryonic stem (ES) cells carrying the conditional mb-1 allele were generated by homologous recombination following standard techniques (Torres and Kuhn 1997). By transiently transfecting these latter cells with a Cre-expressing vector, we isolated ES clones carrying the deletion of the neomycin resistance (neo) cassette and the floxed mb-1 targeted allele in either orientation, thus resulting in the generation of mice carrying either mEGFP/mb-1 or mb-1/mEGFP conditional alleles. A schematic representation and Southern analysis of wild-type and targeted mb-1 alleles are shown in Figure 1a–c. Expression of mEGFP (Fig. 2) and of Ig(data not shown) by the targeted mb-1 alleles was confirmed by flow cytometric analysis of bone marrow, spleen, and lymph node cells isolated from heterozygous mEGFP/mb-1 and homozygous mb-1/mEGFP mice, respectively. The mEGFP/mb-1 allele directs expression of mEGFP, but not Ig. Thus we found that homozygous mEGFP/mb-1 mice lack any Igexpression and their B cell development is absolutely blocked at the B cell progenitor stage (data not shown). On the other hand, the mb-1/mEGFP allele encodes for Ig, but not mEGFP (Fig. 2c and data not shown). The expression of Igby the conditional allele was found to be reduced compared to wild-type (data not shown). As expected and observed in previous cell line experiments, the continuous presence of Cre in the nuclei results in the repetitive inversion (flipping) of the floxed DNA sequence that carries inverted loxP sites (data not shown). Removal of nuclear Cre stops further loxP recombination, stabilizing the floxed DNA into one of the two possible orientations with equal probability. Thus, the resulting cell population contains cells with either orientation of the floxed DNA sequence. The conditional mb-1 mice were tested by breeding them to either polyI/C, (MxCre, Kuhn et al., 1995) or hydroxy-tamoxifen, (MerCreMer, Hobeika et al., unpublished) inducible Cre-transgenic mouse strains. Induction of Cre recombinase in mEGFP/mb-1;MxCre heterozygous mice resulted in the elimination of mEGFP expression in approximately 30% of the bone marrow and splenic B cells (Fig. 2a). Southern blot analysis confirmed the presence of cells carrying mb-1/mEGFP alleles, which were the result of Cre-mediated recombination of mEGFP/mb-1 alleles (Fig. 2b). On the other hand, Cre induction in mb-1/mEGFP;MerCreMer bone marrow cells in vitro resulted in the de novo expression of mEGFP in approximately 9% of B cells (Fig. 2c). Thus, our experiments demonstrated that the mb-1 targeted alleles described here are functional with respect to their capacity to be conditionally activated by inducible Cre recombinase. Conditional mb-1 mice will allow study of the kinetics and functions of B cell development by the induction of Igexpression and the role of Igand BCR signaling in mature B cells, by suppression of Igexpression.

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G.

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

The Glycoprotein gp48 of Murine Cytomegalovirus: PROTEASOME-DEPENDENT CYTOSOLIC DISLOCATION AND DEGRADATION

Degradation of misfolded or unassembled proteins that are co-translationally inserted into the endoplasmic reticulum involves the cytosolic proteasome system. Different principles may exist for the export of proteins into the cytosol for proteasomal degradation. Here we studied the degradation pathway of the viral glycoprotein gp48, a type I transmembrane protein, encoded by the m06 gene of murine cytomegalovirus. In cells stably transfected with the cytomegalovirus m06 gene or infected with the virus itself, two populations of gp48 can be distinguished that have different fates. Complexes of gp48 and the major histocompatibility complex (MHC) class I molecule, are transported to the lysosome for degradation. Unassembled gp48 is degraded by the cytosolic proteasome. Proteasomal inhibitors stabilize the unassembled gp48 in its core-glycosylated and membrane-associated form in the endoplasmic reticulum (ER)-Golgi intermediate compartment. This implicates that both endoplasmic reticulum and ER-Golgi intermediate compartment export gp48 and that degradation is coupled to a functional proteasome. Analysis of gp48 mutants revealed that the cytosolic part of gp48 was not responsible for the proteasome-dependent substrate transport out of the ER-Golgi intermediate compartment. Thus an indirect interaction between the proteasome and its substrate has to be discussed.

Abnormal expression of various molecular forms and distribution of T cell receptor zeta chain in patients with systemic lupus erythematosus.

T cells from the majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain expression. The TCR zeta chain, a critical signaling molecule, exists in multiple molecular forms and membrane fractions with distinct functions in antigen-mediated signaling processes. This study was undertaken to investigate the complete spectrum of expression of the different forms and distribution of the TCR zeta chain in SLE T cells. T cells were isolated from 48 SLE patients and 21 healthy subjects. The expression of various forms of the TCR zeta chain was investigated by immunoblotting with specific antibodies. The lipid raft-associated form of the zeta chain was determined by quantitating the solubilized zeta chain after disruption of the lipid rafts by cholesterol depletion using methyl-betacyclodextrin. The distribution of the zeta chain was investigated by fluorescence microscopy. The phosphorylated 21- and 23-kd forms and the detergent-insoluble membrane-associated form of the TCR zeta chain and alternatively spliced zeta chain were significantly decreased in SLE T cells. In contrast, major ubiquitinated forms of the zeta chain were increased in these cells. We also identified up-regulation of a novel 14-kd form of the zeta chain in SLE T cells. Resting SLE T cell membranes had an increased percentage of the residual membrane-bound zeta chain in the lipid rafts. Fluorescence microscopy findings indicated that the residual zeta chain is more clustered on the cell membranes of SLE T cells. These results suggest that, in addition to the 16-kd form, expression of other molecular forms and fractions of the TCR zeta chain as well as its membrane distribution are abnormal in SLE T cells. Increased lipid raft association and surface clustering of the zeta chain may explain the molecular mechanisms underlying the signaling abnormalities in these cells.

Alpha 1 Connexin (connexin43) gap junctions and activities of cAMP-dependent protein kinase and protein kinase C in developing mouse heart.

alpha 1 Connexin (connexin43) is the dominant gap junction protein of the developing and mature heart where it forms channels that mediate intercellular electrical and metabolic coupling events that are critical for heart function. alpha1 connexin channels are rapidly and reversibly gated by actions of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), and disruption of consensus sites for these phosphorylations are associated with severe heart malformations. However, there have been no reports on the relative activities of PKA or PKC in early heart formation. Nor has the presence and phosphorylation state of alpha1 connexin been documented in these same developmental stages. To begin these studies, we used hearts from 8.5-18.5 dpc (days postcoitus) mouse embryos, postpartum pups, and adults. Membrane or supernatant fractions were used for immunoblots to assess the amounts and distribution of alpha1 connexin protein and each protein kinase. Phosphotransferase assays were done to document the endogenous activities of PKA and PKC. Three species of alpha1 connexin at 44, 46, and 49 kDa were evident in 8.5- and 9.5-dpc embryos and adult hearts, but the 49-kDa band was not consistently found in 10.5 dpc or embryos through 18.5 dpc, although it was robust in adult heart. The amount of PKA was minimal in 8.5-dpc hearts but rose thereafter and was maximal by 10.5 dpc and remained stable throughout development. Catalytic activity of this enzyme was minimal in 8.5-dpc hearts then rose thereafter and was maximal by 10.5 dpc of development. PKC delta was confined mainly to membrane fractions, whereas PKC epsilon had supernatant- and membrane-associated forms. Both enzyme isoforms showed large fluctuations throughout development. In 8.5- and 9.5-dpc hearts, PKC catalytic activity was maximal but, by 10.5 dpc, activity dramatically declined and remained low thereafter. The results demonstrate that alpha1 connexin is present at the heart tube stage (8.5 dpc) of development onward and provide evidence suggesting that channels formed by this protein are dynamically regulated by PKA and PKC, especially in 8.5- and 9.5-day embryonic hearts, which are crucial times for heart formation and left/right patterning in general.

Transient translocation of hemidesmosomal bullous pemphigoid antigen 1 from cytosol to membrane fractions by 12- O-tetradecanoylphorbol-13-acetate treatment and Ca 2+-switch in a human carcinoma cell line

We previously showed that 12- O-tetradecanoylphorbol-13-acetate (TPA) and Ca 2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture medium, which were also linked to activation of protein kinase C (PKC), lead to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but not of 230 kDa-BPAG1, and possibly to its disassembly from hemidesmosomes in a human squamous cell carcinoma cell line (DJM-1). In this study, we examined the effects of TPA and Ca 2+-switch on intracellular localization of BPAG1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal antibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cultured in low Ca 2+ medium, BPAG1 was detected as phosphate buffered saline-soluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca 2+-grown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca 2+-cultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cytosol to membrane fractions within 10 min, that was inhibited by pretreatment with H7 (a selective PKC inhibitor) at 40 μM. After 30 min and 4 h of TPA-treatment, BPAG1 was exclusively detected in cytoskeleton fractions. Morphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear pattern at cell–cell contacts, suggesting translocation of BPAG1 from the cytosol to the plasma membrane. In contrast, the Ca 2+-switch from low to normal caused a prominent increase of BPAG1, both in cytosolic and membrane-associated forms after 4 h, that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 μM, suggesting a role for PKC and BPAG1 synthesis in these Ca 2+-induced effects. These results suggest that TPA and Ca 2+-switch induced BPAG1 translocation to membrane fractions possibly mediated by PKC-activation. Furthermore, whereas TPA affects the redistribution of BPAG1 among their pools without inducing their synthesis, Ca 2+-switch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synthesis.

Detection of the Presenilin 1 COOH-Terminal Fragment in the Extracellular Compartment: A Release Enhanced by Apoptosis

Mutations in gene encoding presenilin 1 (PS1) are responsible for the majority of familial Alzheimer's disease (FAD) cases. We studied PS1 localization in HEK293 cells and in primary neurons obtained from rat cortex and hippocampus. We first demonstrated that PS1-CTF, but neither PS1-FL nor PS1-NTF, is released into the medium as a soluble and membrane-associated form. After induction of apoptosis with staurosporine (Sts), we observed a dramatic increase in the level of PS1-CTF in the medium, both in HEK293 and in primary neurons. Immunocytochemical analysis suggested that the release of PS1-CTF might occur via membrane shedding. Aβ1–42 treatment reduced PS1-CTF extracellular levels. This decrease was strongly associated to an impaired secretion of sAPP fragments, thus suggesting a role of PS1-CTF in the control of trafficking and generation of APP fragments.

Membrane heparan sulfate proteoglycan-supported FGF2-FGFR1 signaling: evidence in support of the "cooperative end structures" model.

Fibroblast growth factor 2 (FGF2)-initiated FGF receptor (FGFR)-signaling requires the assistance of heparin/heparan sulfate. Here, we evaluated the effects of different heparan sulfate proteoglycan (HSPG)-expressing cell lines and HSPGs derived from these cells on FGF2-induced FGFR1-phosphorylation in heparan sulfate-negative BaF3 cells. HSPGs supplied in membrane-associated form, by presenting cells, were all effective promotors of FGF2-initiated FGFR1 phosphorylation, independently of their nature (syndecan/glypican) or cellular origin (human lung fibroblasts, transfected Namalwa cells, or transfected K562 cells). A treatment with heparitinase initially stimulated, but finally completely inhibited, the activity of these presenting cells. In comparison, equivalent amounts of soluble HSPGs, obtained by trypsinization of these cells or by immunopurification from cell extracts, did not promote FGF2-induced FGFR1-phosphorylation, yet removal of the less anionic species or a further treatment with heparitinase converted these soluble fractions into potent activators of FGF2/FGFR1 signaling. Extrapolating from current structural models, we suggest that FGFR dimerization and autophosphorylation is supported by cooperative "heparin-like end structures," and that cell surface association and concentration compensate for the relative scarcity of such end structures in native HSPGs. In this model, "proteolytic" shedding of heparan sulfate would act as a diluting, down-regulatory mechanism, while "heparanolytic" shedding might act as an up-regulatory mechanism, by increasing the concentration of these end structures.

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells.

Membrane-associated binding sites for estrogen may mediate rapid effects of estradiol-17beta that contribute to proliferation of human breast cancers. After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of specific estradiol binding is found in nuclear fractions. However, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes. These estradiol binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei. Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17beta and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of estrogen receptor. Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogen-activated protein kinase and Akt kinase signaling. Estrogen-promoted growth of human breast cancer xenografts in nude mice was also significantly reduced by treatment in vivo with the estrogen receptor antibody. Thus, membrane-associated forms of estrogen receptor may play a role in promoting intracellular signaling for hormone-mediated proliferation and survival of breast cancers and offer a new target for antitumor therapy.

Epidermal growth factor modulates transforming growth factor receptor messenger RNA and protein levels in hamster preantral follicles in vitro.

Epidermal growth factor (EGF) is mitogenic to preantral follicles, and transforming growth factor beta (TGFbeta) influences ovarian cell functions in a variety of species. Although an interaction of these ligands during preantral folliculogenesis is likely, whether EGF influences TGFbeta action on preantral follicles by modulating TGFbeta receptor (TbetaR) gene transcription and translation is not known. To determine whether EGF influenced TbetaR mRNA and protein levels in granulosa cells during preantral folliculogenesis, hamster preantral follicles at stages 1-6 were cultured in the absence or presence of EGF and follicular TbetaR mRNA, and protein levels were monitored by semiquantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. Both TbetaR type I (TbetaRI) and TbetaR type II (TbetaRII) mRNA and protein were present in preantral follicles, and their expression was up-regulated by EGF in a stage-dependent manner. However, EGF effect on the expression of TbetaRI and TbetaRII was differential. In contrast to TbetaRI, EGF-stimulation of follicular TbetaRII mRNA expression was evident from stages 1 and 2 onwards, and more than twofold induction was noted for stages 4-6. Moreover, significant increases in thecal TbetaR mRNA levels were noted for stage 6 follicles. Follicles at smaller stages appeared to be more sensitive to EGF than were larger preantral follicles. Despite an increase in the cytosolic form of TbetaRI protein for most of the stages and TbetaRII protein for follicles at stages 4 and 5, EGF-stimulation of the membrane-associated form of the receptor was restricted to follicles at stage 6. Functionally, TGFbeta1 attenuated EGF-induced DNA synthesis for follicles at stages 1-3 and 6 without affecting EGF-induced progesterone production for most of the stages. Administration of alpha-amanitin resulted in a significant reduction of EGF-induction of TbetaR mRNA levels, suggesting that increased receptor protein levels were a consequence of mRNA synthesis. These results indicate that an interaction between EGF and TGFbeta forms an important regulatory mechanism for preantral folliculogenesis. The effect of EGF on TbetaRI and TbetaRII gene transcription and translation are differential, and follicular response to EGF depends on the developmental status of the follicles.

Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways.

Approximately 50% of metastatic tumors contain Ras mutations. Ras proteins can activate at least three downstream signaling cascades mediated by the Raf-MEK-extracellular signal-regulated kinase family, phosphatidylinositol-3 (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (RalGEFs). Here we investigated the contribution of RalGEF and ERK activation to the development of experimental metastasis in vivo and associated invasive properties in vitro. Each pathway contributes distinct properties to the metastatic phenotype. Following lateral tail vein injection, 3T3 cells transformed by constitutively active Raf or MEK produced lung metastasis that displayed circumscribed, noninfiltrating borders. In contrast, 3T3 cells transformed by Ras(12V,37G), a Ras effector mutant that activates RalGEF but not Raf or P13 kinase, formed aggressive, infiltrative metastasis. Dominant negative RalB inhibited Ras(12V,37G)-activated invasion and metastasis, demonstrating the necessity of the RalGEF pathway for a fully transformed phenotype. Moreover, 3T3 cells constitutively expressing a membrane-associated form of RalGEF (RalGDS-CAAX) formed invasive tumors as well, demonstrating that activation of a RalGEF pathway is sufficient to initiate the invasive phenotype. Despite the fact that Ras(12V,37G) expression does not elevate ERK activity, inhibition of this kinase by a conditionally expressed ERK phosphatase demonstrated that ERK activity was necessary for Ras(12V,37G)-transformed cells to express matrix-degrading activity in vitro and tissue invasiveness in vivo. Therefore, these experiments have revealed a hitherto-unknown but essential interaction of the RalGEF and ERK pathways to produce a malignant phenotype. The generality of the role of the RalGEF pathway in metastasis is supported by the finding that Ras(12V,37G) increased the invasiveness of epithelial cells as well as fibroblasts.

Surface NADH Oxidase of HeLa Cells Lacks Intrinsic Membrane Binding Motifs

Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37°C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis.

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.

GC1q-R/p33, a member of a new class of multifunctional and multicompartmental cellular proteins, is involved in inflammation and infection.

Human gC1q-R (p33, p32, C1qBP, TAP) is a ubiquitously expressed, multiligand-binding, multicompartmental cellular protein involved in various ligand-mediated cellular responses. Although expressed on the surface of cells, an intriguing feature of the membrane-associated form of gC1q-R is that its translated amino acid sequence does not predict the presence of either a sequence motif compatible with a transmembrane segment or a consensus site for a glycosylphosphatidylinositol anchor. Moreover, the N-terminal sequence of the pre-pro-protein gC1q-R contains a motif that targets the molecule to the mitochondria and as such was deemed unlikely to be expressed on the surface. However, several lines of experimental evidence clearly show that gC1q-R is present in all compartments of the cell, including the extracellular cell surface. First, surface labeling of B lymphocytes with the membrane-impermeable reagent sulfosuccinimidyl 6-(biotinamido)hexanoate shows specific biotin incorporation into the surface-expressed but not the intracellular form of gC1q-R. Second, FACS and confocal laser scanning microscopic analyses using anti-gC1q-R IgG mAb 60.11 or 74.5.2, and the fluorophore Alexa 488-conjugated F(ab')2 goat anti-mouse IgG as a probe, demonstrated specific staining of Raji cells (>95% viable). Three-dimensional analyses of the same cells by confocal microscopy showed staining distribution that was consistent with surface expression. Third, endothelial gC1q-R, which is associated with the urokinase plasminogen activator receptor, and cytokeratin 1 bind 125I-high molecular weight kininogen in a specific manner, and the binding is inhibited dose-dependently by mAb 74.5.2 recognizing gC1q-R residues 204-218. Fourth, native gC1q-R purified from Raji cell membranes but not intracellular gC1q-R is glycosylated, as evidenced by a positive periodic acid Schiff stain as well as sensitivity to digestion with endoglycosidase H and F. Finally, cross-linking experiments using C1q as a ligand indicate that both cC1q-R and gC1q-R are co-immunoprecipitated with anti-C1q. Taken together, the evidence accumulated to date supports the concept that in addition to its intracellular localization, gC1q-R is expressed on the cell surface and can serve as a binding site for plasma and microbial proteins, but also challenges the existing paradigm that mitochondrial proteins never leave their designated compartment. It is therefore proposed that gC1q-R belongs to a growing list of a class of proteins initially targeted to the mitochondria but then exported to different compartments of the cell through specific mechanisms which have yet to be identified. The designation 'multifunctional and multicompartmental cellular proteins' is proposed for this class of proteins.