A 60 kDa plasma membrane protein changes its localization to autophagosome and autolysosome membranes during induction of autophagy in rat hepatoma cell line, H-4-II-E cells.

We previously reported the preparation and characterization of an antibody against membrane fraction of autolysosomes from rat liver (J. Histochem. Cytochem. 38, 1571-1581, 1990). Immunoblot analyses of total membrane fraction of a rat hepatoma cell line, H-4-II-E cells by this antibody suggested that H-4-II-E cells expressed several autolysosomal proteins, including a protein with apparent molecular weight of 60 kDa. It was suggested that this 60 kDa protein was a peripheral membrane protein, because it was eluted from the membrane by sodium carbonate treatment. We prepared an antibody against this 60 kDa protein by affinity purification method, and examined its behavior during induction of autophagy. Autophagy was induced by transferring the cells from Dulbecco's modified Eagle medium (DMEM) containing 12% fetal calf serum into Hanks' balance salt solution. In DMEM, the 60 kDa protein showed diffused immunofluorescence pattern, and immunoelectron microscopy suggested that this protein was located on the extracellular side of the plasma membrane. After inducing autophagy, the immunofluorescence configuration of the 60 kDa protein changed from the diffused pattern to a granulous one. Immunoelectron microscopy showed that the 60 kDa protein was localized on the luminal side of the limiting membrane of autolysosomes and endosomes. In the presence of bafilomycin A1 which prevents fusion between autophagosomes and lysosomes, the 60 kDa protein was localized on the limiting membrane of the autophagosomes and endosomes. These results suggest that the 60 kDa protein is transported from the plasma membrane to the autophagosome membrane through the endosomes.     

Purification of a cell growth factor from a human lung cancer cell line: Its relationship with ferritin

We have purified a cell growth factor from a human lung cancer cell line, T3M-30, which was established in a protein-free chemically defined medium. The factor, designated carcinoma-derived growth factor (CD-GF), stimulated proliferation of a variety of cells, including human leukemia cells, HL-60, and melanoma cells, SK-28. Half-maximum stimulation by the purified CD-GF was achieved at a concentration of 40 ng/ml. In the purified CD-GF, two major protein bands of 24 kDa and 22 kDa were identified on a SDS polyacrylamide gel. The partial amino acid sequences of the 24 kDa protein were determined from two peptide fragments obtained by V8 protease treatment. The partial sequences were identical to those of heavy chain of human ferritin. The activity of the purified CD-GF was coprecipitated completely with a monoclonal antibody to heavy chain of ferritin. Ferritin has been considered to inhibit cell growth. However, human heart ferritin was capable of stimulating the growth of HL-60 cells. These results suggest that CD-GF is related to feritin and ferritin is a growth factor of HL-60 leukemia cells. © 1994 Wiley-Liss, Inc.     

Biosynthesis and processing of the mannose receptor in human macrophages

The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.     

Tumor necrosis factor alpha stimulates sphingomyelinase through the 55 kDa receptor in HL-60 cells.

Tumor necrosis factor alpha (TNF alpha) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL-60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNF alpha was concentration dependent with a maximal effect at 3-10 nM. The monoclonal antibody (mAb), htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr-1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNF alpha. In addition, htr-9 or TNF alpha stimulated hydrolysis of sphingomyelin in a membrane fraction of HL-60 cells. These results are consistent with a role of sphingomyelin hydrolysis as an early event in the signalling mechanism of TNF alpha, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.     

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel

The tritiated arylazido phenylalkylamine (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4, 5-trimethoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle transverse-tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a Kd of 2.0 +/- 0.5 nM and a Bmax of 17.0 +/- 0.9 pmol/mg protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high-affinity drug receptor carrying subunit of the skeletal muscle transverse-tubule L-type calcium channel.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

Identification of a dihydropyridine-sensitive calcium channel in chick brain by a monoclonal antibody

A Balb/c mouse was immunized with chick synaptic plasma membranes and monoclonal antibodies were produced by fusion of spleen cells with NS-1 mouse myeloma cells. One antibody, MAC-L1, immunoprecipitated more than 90% of the [3H]PN200-110-labeled calcium channels but only 20% of the omega -conotoxin receptor solubilized from the chick brain membranes. Thus possibly, a certain portion of the omega -conotoxin receptor in the chick brain is a dihydropyridine-sensitive calcium channel. By the specific immunoprecipitation of 125I-labeled proteins, two large polypeptides of 193kDa and 130kDa under reducing conditions were identified as the major components of the calcium channel.     

Detection and localization of a putative cyclic-GMP-activated channel protein in the protozoan ciliate Stentor coeruleus

Summary. Immunoblotting and immunocytochemical assays were employed to identify and localize a channel protein activated by cyclic GMP (cGMP) in the protozoan ciliate Stentor coeruleus. Analysis of whole-cell homogenate with antibodies raised against the α-subunit of the cGMP-activated channel protein from bovine rod outer segments and against cGMP revealed four major protein bands with molecular masses of 40 kDa, 63 kDa, and over 120 kDa, which bound cGMP. However, only a cGMP-binding protein of 63 kDa, corresponding to the α-subunit of the cGMP-activated ion channel protein from bovine rod outer segments, was found in the ciliate cortex fraction. The functional cGMP-activated channel protein was also shown to be present in the cortex fraction of S. coeruleus by patch-clamp measurements of artificial liposomes. Incorporation of the cortex fraction into liposomes resulted in the appearance of ion channel activity related to cGMP. The reconstituted protein channels were strongly inhibited by l-cis-diltiazem, a known potent blocker of many types of cyclic-nucleotide-activated channels. The results presented here are the first demonstration of the existence and localization of a putative cGMP-activated channel protein in the ciliate S. coeruleus. Cyclic-nucleotide-activated channel proteins are nonspecific cation channels which mediate the receptor potentials in photoreceptor cells and in cells of the olfactory epithelium. On the basis of these data, we suggest that the 63 kDa protein identified in Stentor coeruleus is also a cGMP-activated ion channel and that it may be involved as an effector in the photosensory transduction pathway leading to the motile photophobic response in this ciliate protist. Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, 3, Pasteur ulica, 02 093 Warsaw, Poland.     

Progesterone as a regulator of granulosa cell viability

Progesterone (P4) prevents numerous cells, including uterine, mammary and ovarian cells, from undergoing apoptosis. Interestingly, P4 prevents apoptosis of ovarian granulosa cells (GCs), which do not express the classic nuclear P4 receptor. This review presents data that support a non-genomic action of P4 in granulosa cells. These studies were conducted using both primary rat granulosa cells and rat spontaneously immortalized granulosa cells (SIGCs). Specifically, these studies reveal that (1) ³H-P4 specifically binds to SIGCs; (2) an antibody directed against the ligand binding domain of the nuclear P4 receptor (C-262) detects a 60 kDa protein, which localizes to the plasma membrane and binds P4; and (3) treatment with C-262 blocks P4’s ability to maintain granulosa cell viability. Additional studies demonstrate that a protein kinase G (PKG) activator, 8-br-cGMP, mimics and PKG antagonists, Rp-8-pcCPT-GMP and KT5823, attenuate P4’s action. These studies support the concept that the 60 kDa P4 binding protein functions as membrane receptor for P4 which activates a PKG-dependent mechanism to regulate granulosa cell survival.     

Biochemical analysis of a T cell receptor alpha-like molecule involved in antigen-nonspecific suppression

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses a pleiotropic antigen-nonspecific suppressive function. We have shown that 70 kDa MNSF comprises an 8 kDa ubiquitin-like polypeptide (Ubi-L) and 62 kDa T cell receptor (TCR) alpha-like molecule. Ubi-L binds specifically to its 82 kDa receptor protein on target cells. In the current study, we have further characterized the biochemical nature of the TCR(alpha)-like molecule. The 62 kDa protein was separated into two species of 46 kDa and 16 kDa on reverse-phase HPLC. Anti-TCR(alpha) monoclonal antibody recognized the 46 kDa, but not the 16 kDa protein. Anti-TCRbeta monoclonal antibody failed to recognize these proteins. Ubi-L conjugated to the 46 kDa protein, whereas Ubi-L lacking its C-terminal Gly-Gly did not. Although Ubi-L was labile both to heating at 56 degrees C and to acidification to pH 4, the Ubi-L-46 kDa protein complex was unaffected by these treatments. In addition, the 46 kDa protein elongated the Ubi-L-induced protein tyrosine phosphorylation in a concanavalin A-activated murine T helper type 2 clone, D10 cells. One of the four tryptic peptide sequences derived from the 46 kDa protein was in alignment with a related sequence found in the J(alpha) region of the TCR(alpha), including the highly conserved motif F-G-X-G-T-X-L.     

Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum.

The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T-tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization-induced Ca2+ release nor on caffeine-induced Ca2+ release. Since the slow phase of depolarization-induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Rapid actions of progesterone on granulosa cells

Ovarian granulosa cells are responsive to progesterone but do not express the nuclear progesterone receptor. In an attempt to identify a receptor for progesterone (P4) in granulosa cells (GCs), an antibody built against the ligand binding site of the P4 receptor (i.e. C-262) was used. This antibody detected a 60 kDa protein in GCs as well as spontaneously immortalized granulosa cells (SIGCs). This C-262 detectable protein localizes to the plasma membrane and binds P4. Importantly, this C-262 detectable protein appears to be involved in mediating P4's biological actions. This is based on the findings that C-262 1) blocks P4's ability to inhibit mitogen-induced mitosis and apoptosis and 2) FITC-BSA-conjugated P4 binding to granulosa cells. A C-262 detectable protein was isolated using a C-262 affinity column and sequenced. This analysis identified an unnamed protein referred to as RDA288 that was found in the rat genome (Accession number: XM216160). A nearly identical unnamed protein was found in a cDNA library of mouse lung (Accession number: AK004678). Whether RDA288 functions as a membrane receptor for P4 remains to be determined.     

Human placental sialidase complex: characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies

Sialidase isolated from human placenta is associated with several proteins including acid beta-galactosidase, carboxypeptidase, N-acetyl-alpha-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449-456, 1990) as a sialidase protein: this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (gamma 1).     

Isolation and characteristics of monoclonal antibodies to the external domain of the EGF receptor in human A431 epidermoid carcinoma

The monoclonal antibody to the epidermal growth factor (EGF) receptor was generated after fusion of PAI myeloma cells with immunized BALB/c mouse spleen cells, using intact A431 epidermoid carcinoma cells as an immunogen. The antibody, denoted 5A9, is an IgG, which recognizes a protein with molecular mass 170 kDa during immunoblot analysis, immunoprecipitates phosphoprotein with molecular mass 170 kDa from the membrane preparations of A431 cells, and, according to immunofluorescence experiments, is distributed in the cell similar to the EGF-rhodamine conjugate. It is concluded that the produced antibodies are specific to EGF-receptor. At the same time the 5A9 (50 nM) do not compete with EGF for binding with high and low affinity receptors. They fail to induce internalization of the EGF-receptor and do not exert influence on intracellular degradation of EGF-receptor. Monoclonal antibodies 5A9 are also unable to inhibit the EGF-induced protein kinase activity of the receptor and do not stimulate protein kinase activity by themselves. Thus, the prepared monoclonal antibodies can be used to register the EGF-receptor cellular localization without affecting biologic activity of the receptor.     

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.