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W H Moolenaar

Publications and source records attributed to W H Moolenaar.

At least 55 records · Page 3Linked to original sources
PubMed1996

Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor.

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.

Animals↗
PubMed1996

Lack of association between receptor protein tyrosine phosphatase RPTP mu and cadherins.

RPTP mu is a receptor-like protein tyrosine phosphatase that mediates homophilic cell-cell interactions. Surface expression of RPTP mu is restricted to cell-cell contacts and is upregulated with increasing cell density, suggesting a role for RPTP mu in contact-mediated signaling. It was recently reported (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977-986) that RPTP mu binds directly to cadherin/catenin complexes, and thus may regulate the tyrosine phosphorylation of such complexes. Here we report that this concept needs revision. Through reciprocal precipitations using a variety of antibodies against RPTP mu, cadherins, and catenins, we show that RPTP mu does not interact with cadherin/catenin complexes, even when assayed under very mild lysis conditions. We find that the anti-RPTP mu antiserum used by others precipitates cadherins in a nonspecific manner independent of RPTP mu. We conclude that, contrary to previous claims, RPTP mu does not interact with cadherin complexes and thus is unlikely to directly regulate cadherin/catenin function.

Animals↗
PubMed1995

Homophilic interactions mediated by receptor tyrosine phosphatases mu and kappa. A critical role for the novel extracellular MAM domain.

The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTP mu and RPTP kappa can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTP mu and RPTP kappa fail to interact in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTP mu lacking the MAM domain in insect Sf9 cells. Truncated RPTP mu is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored in a chimeric RPTP mu molecule containing the MAM domain of RPTP kappa. However, this chimeric RPTP mu does not interact with either RPTP mu or RPTP kappa. These results indicate that the MAM domain of RPTP mu and RPTP kappa is essential for homophilic cell-cell interaction and helps determine the specificity of these interactions.

Animals↗
PubMed1995

Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells: structure-activity analysis.

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells, which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered, the rank order of potency is oleoyl-LPA > arachidonoyl-LPA > linolenoyl-LPA > linoleoyl-LPA > stearoyl-LPA = palmitoyl-LPA > myristoyl-LPA. The shorter-chain species, lauroyl- and decanoyl-LPA, show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses > 15 microM. Similarly, the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate, a putative messenger with some structural similarities to LPA, elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however, cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that, although many features of the LPA structure are important for optimal activity, the phosphate group is most critical, suggesting that this moiety is directly involved in receptor activation.

Animals↗
PubMed1995

C-terminal truncation of the neurokinin-2 receptor causes enhanced and sustained agonist-induced signaling. Role of receptor phosphorylation in signal attenuation.

The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Ca2+ remain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.

Amino Acid Sequence↗
PubMed1995

Lysophosphatidic acid signalling.

Lysophosphatidic acid is an intercellular phospholipid messenger that is released from platelets (and probably other cells) and evokes multiple biological responses, ranging from induction of mitogenesis to neurite retraction, by activating a specific G protein coupled receptor. Recent studies indicate that the lysophosphatidic acid receptor acts via the small GTP-binding proteins Ras and Rho to stimulate cell proliferation and to trigger actin-based cytoskeletal events, respectively.

Animals↗
PubMed1995

Cell surface expression of receptor protein tyrosine phosphatase RPTP mu is regulated by cell-cell contact.

RPTP mu is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTP mu mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTP mu may function as a cell contact receptor in mink lung epithelial cells, which express RPTPmu endogenously, as well as in transfected 3T3 cells. We find that RPTP mu has a relatively short half-life (3-4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPmu is restricted to regions of tight cell-cell contact. RPTPmu surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTP mu is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPmu surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTP mu function: In the absence of cell-cell contact, newly synthesized RPTP mu molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPmu to be trapped at the surface through homophilic binding, resulting in accumulation of RPTP mu at intercellular contact regions. This contact-induced clustering of RPTPmu may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.

3T3 Cells↗
PubMed1994

Phosphatidic acid activation of protein kinase C-zeta overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids.

Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.

Animals↗
PubMed1994

cAMP abrogates the p21ras-mitogen-activated protein kinase pathway in fibroblasts.

The mechanism by which cAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not understood. Here we show that in Rat-1 fibroblasts, cAMP-raising agents inhibit p21ras-mediated mitogen-activated protein (MAP) kinase activation induced by either epidermal growth factor or lysophosphatidic acid. Under the same conditions, however, epidermal growth factor- or lysophosphatidic acid-induced protein tyrosine phosphorylation, Ca2+ mobilization, and activation of Na+/H+ exchange are not attenuated. In ras-transformed Rat-1 cells, 8-bromo-cAMP rapidly deactivates constitutively active MAP kinase without reducing p21ras.GTP levels; long term 8-bromo-cAMP treatment of these cells leads to growth arrest and reversion of the transformed phenotype. These results show that elevation of intracellular cAMP levels abrogates the p21ras MAP kinase pathway at a step downstream of p21ras activation. This finding provides a molecular basis for the growth-inhibitory action of cAMP in normal and transformed fibroblasts.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway.

Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21ras through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21ras and to stimulate DNA synthesis in these cells. LPA and, more effectively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p125FAK) but not the 120-kDa p21ras GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the p42/p44 mitogen-activated protein (MAP) kinases, paralleling p21ras activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 microM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21ras and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21ras activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21ras, and tyrosine phosphorylation of the other substrates, including p125FAK, is associated with phospholipase C activation.

Adenylate Cyclase Toxin↗
PubMed1994

LPA: a novel lipid mediator with diverse biological actions.

Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, is a platelet-derived serum factor that evokes a wide range of biological effects, including stimulation of fibroblast proliferation, platelet aggregation, cellular motility, tumour cell invasiveness and neurite retraction. This review summarizes recent insights into the mode of action of LPA. LPA appears to activate its own G-protein-coupled receptor(s) to initiate both classic and novel signal cascades. Of particular interest is LPA's ability to activate the Ras pathway and to stimulate protein tyrosine phosphorylation in concert with remodelling of the actin cytoskeleton.

Journal Article↗
PubMed1994

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

ADP Ribose Transferases↗
PubMed1993

Purification and characterization of the cytoplasmic domain of human receptor-like protein tyrosine phosphatase RPTP mu.

RPTP mu is a recently described receptor-like protein tyrosine phosphatase (PTP), the ectodomain of which mediates homophilic cell-cell adhesion. The cytoplasmic part contains two homologous PTP-like domains and a juxtamembrane region that is about twice as large as in other receptor-like PTPs. The entire 80-kDa cytoplasmic part of human RPTP mu was expressed in insect Sf9 cells and its enzymatic activity was characterized after purification to electrophoretic homogeneity. In addition, the effects of deletion and point mutations were analyzed following expression in Escherichia coli cells. The purified cytoplasmic part of RPTP mu displays high activity toward tyrosine-phosphorylated, modified lysozyme (Vmax 4500 nmol min-1 mg-1) and myelin basic protein (Vmax 8500 nmol min-1 mg-1) but negligible activity toward tyrosine-phosphorylated angiotensin or the nonapeptide, EDNDpYINASL, that serves as a good substrate for protein tyrosine phosphatase PTP1B. This suggests that RPTP mu and PTP1B have distinct substrate specificities. Catalytic activity is independent of Ca2+ (up to 1 mM) but is strongly inhibited by Zn2+, Mn2+, vanadate, phenylarsenic oxide, and heparin. The first of the two catalytic domains is 5-10 times less active than the expressed catalytic region containing both domains. Mutation of Cys 1095 to Ser in the first catalytic domain abolishes enzymatic activity when analyzed following expression in either E. coli or mammalian COS cells. Deletion of the first 53 amino acids from the juxtamembrane region reduces catalytic activity about 2-fold.

Animals↗
PubMed1993

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Animals↗
PubMed1993

Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase.

Receptor-like protein tyrosine phosphatases (receptor-PTPs) represent a novel family of transmembrane proteins that are thought to play important roles in cellular regulation. They consist of a cytoplasmic catalytic region, a single transmembrane segment and an extracellular, putative ligand-binding domain, but the nature of their physiological ligands is unknown. We have recently cloned a new receptor-PTP (RPTP mu), the ectodomain of which includes an Ig-like and four fibronectin type III-like domains, suggesting that RPTP mu may be involved in cell-cell or cell-matrix interactions. To test this hypothesis, we expressed RPTP mu in insect Sf9 cells using recombinant baculovirus. We demonstrate that RPTP mu dramatically promotes cell-to-cell adhesion in a homophilic, Ca(2+)-independent manner. No adhesion is observed in Sf9 cells expressing a chimeric RPTP mu molecule containing the extracellular domain of the epidermal growth factor receptor. Furthermore, cells expressing an enzymatically inactive, point-mutated RPTP mu or a truncated form of RPTP mu, lacking the entire catalytic region, show adhesive properties indistinguishable from those of wild-type RPTP mu, indicating that the catalytic domain is not essential for RPTP mu-mediated adhesion. These results assign a physiological role for RPTP mu in signaling cell-cell recognition.

Animals↗
PubMed1993

The bioactive phospholipid lysophosphatidic acid is released from activated platelets.

Lysophosphatidic acid (LPA) is a water-soluble phospholipid with hormone-like and growth-factor-like activities. LPA activates a putative G-protein-coupled receptor in responsive cells, but the natural source of exogenous LPA is unknown. Here we show that LPA is present in mammalian serum in an active form (bound to albumin) at concentrations of 1-5 microM, but is not detectable in platelet-poor plasma, suggesting that LPA is produced during blood clotting. We find that thrombin activation of platelets prelabelled with [32P]Pi results in the rapid release of newly formed [32P]LPA into the extracellular environment. We conclude that LPA is a novel platelet-derived lipid mediator that may play a role in inflammatory and proliferative responses to injury.

Blood Platelets↗