Showing papers in "Molecular Biology of the Cell in 1991"

Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

Abstract: We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.

Diversity of the cadherin family: evidence for eight new cadherins in nervous tissue.

Abstract: To examine the diversity of the cadherin family, we isolated cDNAs from brain and retina cDNA preparations with the aid of polymerase chain reaction. The products obtained included cDNAs for two of three known cadherins as well as eight distinct cDNAs, of which deduced amino acid sequences show significant similarity with the known cadherin sequences. Larger cDNA clones were isolated from human cDNA libraries for six of the eight new molecules. The deduced amino acid sequences show that the overall structure of these molecules is very similar to that of the known cadherins, indicating that these molecules are new members of the cadherin family. We have tentatively designated these cadherins as cadherin-4 through -11. The new molecules, with the exception of cadherin-4, exhibit features that distinguish them as a group from previously cloned cadherins; they may belong to a new subfamily of cadherins. Northern blot analysis showed that most of these cadherins are expressed mainly in brain, although some are expressed in other tissues as well. These findings show that the cadherin family of adhesion molecules is much larger than previously thought, and suggest that the new cadherins may play an important role in cell-cell interactions within the central nervous system.

Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies.

Abstract: A protein kinase characterized by its ability to phosphorylate microtubule-associated protein-2 (MAP2) and myelin basic protein (MBP) is thought to play a pivotal role in the transduction of signals from many receptors in response to their ligands. A kinase with such activity, named extracellular signal-regulated kinase 1 (ERK1), is activated rapidly by numerous extracellular signals, requires phosphorylation on tyrosine to be fully active, and in vitro can activate a kinase (a ribosomal S6 protein kinase) that is downstream in phosphorylation cascades. From the protein sequence predicted by the rat ERK1 cDNA, peptides were synthesized and used to elicit antibodies. The antibodies recognize both ERK1; a closely related kinase, ERK2; and a third novel ERK-related protein. Using these antibodies we have determined that ERK1 and ERK2 are ubiquitously distributed in rat tissues. Both enzymes are expressed most highly in brain and spinal cord as are their mRNAs. The third ERK protein was found in spinal cord and in testes. The antibodies detect ERKs in cell lines from multiple species, including human, mouse, dog, chicken, and frog, in addition to rat, indicating that the kinases are conserved across species. ERK1 and ERK2 have been separated by chromatography on Mono Q. Stimulation by insulin increases the phosphorylation of both kinases on tyrosine residues, as assessed by immunoblotting with phosphotyrosine antibodies, and retards their elution from Mono Q. Each of these ERKs appears to account for a distinct peak of MBP kinase activity. The activity in each peak is diminished by incubation with either phosphatase 2a or CD45. Therefore, both enzymes have similar modes of regulation and appear to contribute to the growth factor-stimulated MAP2/MBP kinase activity measured in cell extracts.

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite

Abstract: Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.

Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes.

Abstract: Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. We have compared the intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha in two different cell systems. We found that TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha in contrast to the mostly degraded EGF. In addition, TGF-alpha did not induce a complete down-regulation of cell surface receptors, as observed with EGF, which is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation, in the case of TGF-alpha. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF.

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Abstract: Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated. The integrin alpha 6 beta 4 is expressed along the basal surface of various epithelial cells. We show here that this integrin localizes to hemidesmosomes as determined by immunoelectron microscopy using antibodies directed against both the extra- and intracytoplasmic domains of alpha 6 beta 4. This result, which agrees with a recent study, suggests a functional role for the alpha 6 beta 4 integrin in the hemidesmosomes. We therefore investigated such a potential role for this integrin using the cultured rat bladder carcinoma cell line 804G, which has the uncommon ability to form hemidesmosomes in vitro when maintained on uncoated glass substrates. By immunoprecipitation and immunofluorescence, we show that 804G cells express alpha 6 beta 4 along their basal surface in a punctate pattern that overlaps with the distribution of hemidesmosomal plaque antigens. However, this pattern is altered when cells are plated in the presence of an antiserum directed against alpha 6 beta 4. Furthermore, no hemidesmosomes are detectable at the ultrastructural level in the alpha 6 beta 4 antibody-treated cells compared with control cells. These results indicate that integrins may play a critical role in assembly and adhesive functions of the hemidesmosome.

Molecular cloning of a second form of rac protein kinase.

Abstract: A novel serine/threonine protein kinase (termed rac-PK) has recently been identified and cloned from cDNA libraries derived from the human cell lines MCF-7 and WI38. A second form of this protein kinase, termed rac protein kinase beta, has been identified from cDNAs derived from the same cell lines. These two closely related forms show 90% homology, although the beta form with a predicted Mr 60,200 has a carboxyl terminal extension of 40 amino acids in comparison to the alpha form. This extension has a high serine content with 11 serine residues in the last 30 amino acids. The beta form of the protein has been shown by both in vitro translation and bacterial expression to be approximately 5000 Da larger than the alpha form. rac protein kinase beta is encoded by a 3.4-kb transcript and the alpha form is encoded by a 3.2-kb mRNA. Using gene-specific probes both transcripts were detected in all cell types analyzed, although levels of expression were different for the two forms. The catalytic domain of rac protein kinase beta shows a high degree of homology to both the protein kinase C and cyclic AMP-dependent protein kinase families, and hence rac protein kinases appear to represent a new subfamily of the second messenger serine/threonine protein kinases.

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Abstract: Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.

Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells.

Abstract: Transformation of cultured chick lens epithelial cells with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) leads to radical changes in cell shape and interactions. When cultured at the restrictive temperature (42 degrees C), the transformed cells largely retained epithelial morphology and intercellular adherens junctions (AJ), whereas on switch to the permissive temperature (37 degrees C) they rapidly became fibroblastoid, their AJ deteriorated, and cell adhesion molecules (A-CAM) (N-cadherin) largely disappeared from intercellular contact sites. The microfilament system that was primarily associated with these junctions was markedly rearranged on shift to 37 degrees C and remained associated mainly with cell-substrate focal contacts. These apparent changes in intercellular AJ were not accompanied by significant alterations in the cellular content of several junction-associated molecules, including A-CAM, vinculin, and talin. Immunolabeling with phosphotyrosine-specific antibodies indicated that both cell-substrate and intercellular AJ were the major cellular targets for the pp60v-src tyrosine-specific protein kinase. It was further shown that intercellular AJ components serve as substrates to tyrosine kinases also in nontransformed lens cells, because the addition of a combination of vanadate and H2O2--which are potent inhibitors of protein tyrosine phosphatases--leads to a remarkable accumulation of immunoreactive phosphotyrosine-containing proteins in these junctions. This finding suggests that intercellular junctions are major sites of action of protein tyrosine kinases and that protein tyrosine phosphatases play a major role in the regulation of phosphotyrosine levels in AJ of both normal and RSV-transformed cells.

Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

Abstract: In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

Abstract: An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.

Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.

Abstract: The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Abstract: Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.

Laminin-binding integrin alpha 7 beta 1: functional characterization and expression in normal and malignant melanocytes.

Abstract: A novel integrin, alpha 7 beta 1, that specifically binds with high affinity to laminin has been identified on melanoma cells. This complex was purified from both human and murine melanoma cells by laminin-affinity chromatography, and the alpha 7 subunit was recovered after gel electrophoresis. N-terminal amino acid sequence analysis of the alpha 7 subunit from both human and mouse cells verifies that this integrin is distinct from other alpha chains in the beta 1 family, although strikingly similar to the alpha 6 subunit. By using specific proteolytically derived fragments of laminin, it was determined that the alpha 7 beta 1 complex binds selectively to the E8 region, which represents part of the long arm of laminin. In contrast, the receptor failed to bind to the P1 fragment, which contains the intersection of the short arms of laminin. Although the alpha 7 beta 1 complex was commonly expressed in melanoma cells, this integrin was not detected in normal melanocytes, suggesting that alpha 7 expression may be associated with malignant transformation. These results establish the existence of a novel integrin that binds to the E8 domain of laminin and appears to mediate cell adhesion to this ligand.

Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.

Abstract: Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

Abstract: 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.

p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.

Abstract: Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidl...

Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

Abstract: Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes.

Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells.

Abstract: The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 18-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF.

Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

Abstract: Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin. In fact, RA-treated SY5Y cells elongated neurites on laminin-coated substratum more efficiently compared with untreated cells or cells treated with nerve growth factor, insulin, or phorbol 12-myristate 13-acetate. These three agents induced partial morphological differentiation but did not increase alpha 1 integrin expression. Neurite extension in RA-treated cells was more efficient on laminin than on fibronectin or collagen type I and was inhibited with beta 1 integrin antibodies on all three substrates. Affinity chromatography experiments showed that alpha 1/beta 1 is the major laminin receptor in both untreated and RA-treated SY5Y cells. These data show that RA, a naturally occurring morphogen implicated in embryonic development, can selectively regulate the expression of integrin complexes in neuronal cells and suggest an important role of the alpha 1/beta 1 laminin receptor in the morphological differentiation of nerve cells.

Primary structure of a molluscan egg-specific NADase, a second-messenger enzyme.

Abstract: An egg-specific NADase has been purified from the ovotestis of the marine mollusk Aplysia californica. The enzyme converts NAD to cyclic ADP-ribose (cADPR), which is a potent mobilizer of Ca2+. It is likely that the NADase serves to raise Ca2+ levels in the ova at appropriate times. A 1.2-kb cDNA clone containing the complete coding sequence of the native NADase protein was isolated from an unamplified ovotestis cDNA library and represents the first cloning of an NADase that generates cADPR. In vitro translation studies indicate that the protein initially has a signal sequence that may help to target it to discrete vesicles of the ova in which it is found. There are 12 cysteines in the open reading frame, two of these being in the signal sequence. No part of the sequence has significant similarity to other proteins or known nucleotide binding site consensus sequences. Northern blot analysis of poly(A)+ selected ovotestis RNA has identified an NADase mRNA of 1.85 kb. In situ hybridization analysis of cryostat sections from ovotestis has shown that the NADase mRNA is restricted to the immature ova, although the NADase protein is present in both immature and mature eggs.

Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.

Abstract: Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.

Identification of chicken embryo kinase 5, a developmentally regulated receptor-type tyrosine kinase of the Eph family.

Abstract: Chicken embryo kinase 5 (Cek5) is a transmembrane tyrosine kinase of the Eph family that was identified by screening a 10-d chicken embryo cDNA expression library with anti-phosphotyrosine antibodies. The extracellular region of Cek5 contains a cysteine rich N-terminal subdomain and a C-terminal subdomain mostly devoid of cysteines and comprising two repeats similar to fibronectin type III repeats. Immunoblotting experiments with anti-Cek5 polyclonal antibodies indicated that Cek5 is a membrane-associated 120-kDa protein containing intramolecular (but not intermolecular) disulfide bonds. Cek5 is already expressed in 2-d-old chicken embryos and is also expressed, at higher levels, later in development. In 10-d-old chicken embryos, Cek5 is expressed at substantial levels in nearly all the tissues examined, whereas in adult it is expressed predominantly in the brain. The expression of Cek5 in the brain gradually diminishes during embryonic development, whereas in the skeletal muscle of the thigh a sharp decrease in Cek5 expression was detected at the time of terminal muscle differentiation. Its wide tissue distribution throughout development and its sustained expression in adult brain suggest that Cek5 is an important component of signal transduction pathways, likely to interact with a widely distributed and important ligand, which is as yet unknown.

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Abstract: Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.

Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe.

Abstract: The adenylyl cyclase gene, cyr1, of Schizosaccharomyces pombe has been cloned. We have begun an analysis of the function and regulation of adenylyl cyclase by disrupting this gene and by over-expressing all or parts of this gene in various strains. cyr1- strains are viable and contain no measurable cyclic AMP. They conjugate and sporulate under conditions that normally inhibit wild-type strains. Strains containing the cyr1 coding sequences transcribed from the strong adh1 promoter contain greatly elevated adenylyl cyclase activity, as measured in vitro, but only modestly elevated cAMP levels. Such strains conjugate and sporulate less frequently than wild-type cells upon nutrient limitation. Strains which carry the wild-type cyr1 gene but that also express high levels of the amino terminal domain of adenylyl cyclase behave much like cyr1-strains, suggesting that the amino terminal domain can bind a positive regulator. A protein that copurifies with the adenylyl cyclase of S. pombe cross-reacts to antiserum raised against the S. cerevisiae adenylyl cyclase-associated regulatory protein, CAP.

Complementation between urokinase-producing and receptor-producing cells in extracellular matrix degradation.

Abstract: The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.

Steroid-induced epithelial-fibroblastic conversion associated with syndecan suppression in S115 mouse mammary tumor cells.

Abstract: Cell-matrix interactions play an important role in the maintenance of cell shape, supposed to be mediated by the anchorage of cellular cytoskeleton to extracellular matrix via matrix receptors. In this work the expression of one of the known matrix receptors, syndecan, was studied during the hormone-induced change in the phenotype of Shionogi 115 (S115) mouse mammary tumor cells. In the presence of testosterone, when S115 cells express fibroblastic phenotype, they increased their growth rate and became gradually anchorage independent. These cells, however, revealed strong RGDS-dependent binding to fibronectin (FN) but not binding to the heparin-binding domain of FN. Instead, S115 cells growth without testosterone showed epithelial morphology and binding to the heparin-binding domain of FN, suggesting an alteration of syndecan expression in hormone-treated S115 cells. As quantitated by radioimmunoassay and by Western blot, the amounts of both matrix-binding ectodomain of syndecan and syndecan mRNA (2.6 kb) declined in hormone-treated S115 cells. The addition of antiandrogen cyproterone acetate to culture medium opposed the effect of testosterone on syndecan mRNA. We thus propose that the inactivation of syndecan gene and the consequent suppression of syndecan expression is related to the altered adhesion properties, the disappearance of epithelial phenotype, and, on the other hand, to the appearance of transformed-like phenotype in hormone-treated S115 cells.

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

Abstract: A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Intracellular signaling of transcription and secretion of type IV collagen after angiotensin II-induced cellular hypertrophy in cultured proximal tubular cells.

Abstract: Physiologic concentrations of angiotensin II (AII) can induce cellular hypertrophy in murine proximal tubular epithelium (MCT cells). This response is characterized by an increase in cell size, new protein synthesis, and by the secretion of new basement membrane type IV collagen in the absence of cellular proliferation. The present study was undertaken to evaluate the second messengers of these AII-induced cellular events with special reference to the increase in type IV collagen secretion. In initial experiments we observed that pretreatment of MCT cells with agents that increase concentrations of intracellular cAMP, like forskolin, dibutyryl cAMP, and isobutyl-methyl-xanthine abolish AII-induced amino acid incorporation, but have no effect on control cells or on their proliferation. In addition, 10(-8) M AII significantly decreased the concentration of intracellular cAMP. Phorbolesters were without significant effect on the hypertrophy or proliferation of AII-stimulated MCT cells or their rested controls. The transfection of MCT cells with reporter genes containing regulatory elements for type IV collagen revealed that the stimulatory effects of AII on collagen type IV depend, at least to some extent, on an increase in gene transcription. Agents increasing intracellular cAMP concentrations inhibited the AII-induced increase in transcription and secretion of collagen type IV, but had no effect on MCT cells grown in media without AII. Our findings provide evidence that AII-induced changes in tubular epithelium leading to the secretion of type IV collagen are mediated by a decrease in intracellular cAMP.