©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lysophosphatidic Acid, a Multifunctional Phospholipid Messenger (*)

Wouter H. Moolenaar (§)

From the (1) Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

INTRODUCTION
Biochemistry of LPA
Biological Responses to LPA
Structure-Activity Relationship
G Protein-mediated Signal Transduction
Concluding Remarks
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

Research into phospholipid signaling continues to flourish, as more and more bioactive lipids are being identified and their actions characterized. One interesting addition to the growing list of lipid messengers is lysophosphatidic acid (LPA() ; 1-acyl-glycerol-3-phosphate), the simplest of all glycerophospholipids. While LPA has long been known as a precursor of phospholipid biosynthesis in both eukaryotic and prokaryotic cells, only recently has LPA emerged as an intercellular signaling molecule that is rapidly produced and released by activated cells, notably platelets, to influence target cells by acting on a specific cell-surface receptor (1) . Although its precise physiological (and pathological) functions in vivo remain to be explored, LPA derived from platelets has all the hallmarks of an important mediator of wound healing and tissue regeneration. Thus, in addition to acting as an autocrine stimulator of platelet aggregation (1) , LPA stimulates the growth of fibroblasts (1, 2, 3, 4) , vascular smooth muscle cells (5) , endothelial cells,() and keratinocytes (6) ; furthermore, it promotes cellular tension (7) and cell-surface fibronectin binding (8) , which are important events in wound repair. As a product of the blood-clotting process, LPA is a normal constituent of serum (but not platelet-poor plasma), where it is present in an albumin-bound form at physiologically relevant concentrations (9, 10) . Cellular responses to LPA in fact show a striking overlap with those to serum, and it therefore seems safe to conclude that albumin-bound LPA accounts for much of the platelet-derived biological activity of serum.

It is now well established that LPA acts on its cognate G protein-coupled receptor(s), apparently present in many different cell types. Radioligand binding studies have revealed the presence of specific LPA binding sites in membranes from 3T3 cells and rat brain, with K values in the lower nanomolar range (11) . Moreover, cross-linking experiments have identified a candidate high affinity LPA receptor with an apparent molecular mass of 38-40 kDa in LPA-responsive cells and mammalian brain (12) . Although to date a functional LPA receptor has not been purified or cloned, much has already been learned about LPA receptor signaling. The LPA receptor couples to multiple independent effector pathways in a G protein-dependent manner; these include not only ``classic'' second messenger pathways, such as stimulation of PLC and inhibition of adenylyl cyclase, but also novel routes, notably activation of the small GTP-binding proteins Ras and Rho. This minireview aims to introduce the reader to our current understanding of LPA as an intercellular messenger.


Biochemistry of LPA

Despite its ubiquity and physiological importance, relatively little is known about the physical chemistry of LPA such as its phase behavior and cation binding properties. Having a free hydroxyl and phosphate moiety, LPA (oleoyl) is much more water-soluble than other long chain phospholipids (13) . Because of its hydrophilicity LPA does not necessarily remain membrane-associated after its formation and, furthermore, readily escapes detection in conventional lipid extraction procedures. Unlike other lysophospholipids, LPA is not lytic to cells under physiological conditions because of its relatively small headgroup. Calcium ions tend to precipitate LPA from aqueous solutions, which can be prevented by addition of albumin (13) . However, where examined, the biological effects of LPA (see below) are not critically dependent on the presence of albumin.

Production and Release

Besides being synthesized and processed to more complex phospholipids in the endoplasmic reticulum, LPA can be generated through the hydrolysis of pre-existing phospholipids following cell activation. The best documented example concerns thrombin-activated platelets, where LPA production is followed by its extracellular release (10) . Platelet LPA is formed, at least in large part, through PLA-mediated deacylation of newly generated PA (14) , as depicted schematically in Fig. 1 . Distinct PA-specific PLA activity has been identified in platelets (Ca-dependent (15) ) and in rat brain (Ca-independent (16) ), but little is known about its mode of regulation. It remains to be examined at what stage of the platelet activation response LPA is produced and how it is released into the extracellular environment. Given the wide variety of LPA-responsive cell types, LPA production and release are unlikely to be restricted to just platelets. Indeed, there is preliminary evidence that growth factor-stimulated fibroblasts can also produce LPA (17) . Furthermore, LPA may be formed and released by injured cells, probably due to nonspecific activation of phospholipases,() but many other cell systems remain to be examined for LPA production.


Figure 1: Proposed generation of LPA from newly formed PA in activated cells, notably thrombin-stimulated platelets. Diacylglycerol is directly formed via receptor-linked activation of PLC and is then phosphorylated by diacylglycerol kinase to yield PA. PA may also be directly formed through activation of PLD acting on phosphatidylcholine and phosphatidylethanolamine (R denotes phospholipid headgroup). In activated platelets, PA is thought to be hydrolyzed by a specific PLA to yield bioactive LPA (see text). The possible contribution of other pathways to generating LPA remains to be examined.



LPA and LPA-like Lipids in Serum

In freshly prepared mammalian serum, LPA concentrations are estimated to be in the range of approximately 2-20 µM, with oleoyl- and palmitoyl-LPA being the predominant species (5, 10) . LPA is not detectable in platelet-poor plasma, whole blood, or cerebrospinal fluid (9, 10). In common with long chain fatty acids, LPA binds with high affinity to serum albumin at a molar ratio of about 3:1 (18) . It is notable that serum albumin contains several other, as yet unidentified lipids (methanol-extractable) with LPA-like biological activity (9) . This raises the interesting possibility that LPA may belong to a new family of phospholipid mediators showing overlapping biological activities and acting on distinct receptors; conceivably, the ether-linked phospholipid platelet-activating factor (PAF) and the mitogenic lipid sphingosine 1-phosphate (19)() may also belong to this putative family.

LPA Catabolism

Besides binding to its cell-surface receptor(s), LPA is degraded by intact cells. In fibroblasts, exogenous LPA is rapidly hydrolyzed in part to monoacylglycerol and, to a much lesser extent, diacylglycerol and PA (20) . Most of the monoacylglycerol formed remains cell-associated and is presumably further metabolized. Ectophosphohydrolase activity toward LPA has recently been identified in keratinocytes (21) , and an LPA-specific phospholipase has been purified from rat brain (22) . Enzymatic degradation of LPA to biologically inactive products may serve to limit the duration and magnitude of LPA's cellular effects.


Biological Responses to LPA

The list of biological responses to LPA () is quite diverse, ranging from induction of cell proliferation (1, 2, 3, 4, 5, 6) to stimulation of neurite retraction (9, 23) and even slime mold chemotaxis (24) , and continues to grow as more and more cellular systems are being tested for LPA responsiveness (see also Refs. 1 and 2). Where studied, the effects of LPA are highly specific and appear to be receptor-mediated. A caveat is that receptor involvement has not in every case been tested thoroughly. In general, with bioactive lipids such as LPA it is very important to test the formal possibility that ``nonspecific'' incorporation into membranes and/or cellular uptake might somehow influence cell behavior in a receptor-independent manner. Indeed, there is precedent for a phospholipid mediator, notably PAF, to evoke both receptor-dependent and receptor-independent effects (25) . On the other hand, receptor-independent responses to LPA have not been documented thus far.

The mitogenic activities of LPA have been best studied in fibroblasts. As with peptide growth factors, induction of DNA synthesis by LPA requires its long term presence in the medium (1, 2, 3, 4, 26) . The mitogenic action of LPA is almost completely inhibited by PTX, implicating the critical involvement of one or more G-type heterotrimeric G proteins. LPA also stimulates the growth of mouse preimplantation embryos in a PTX-sensitive manner (27) , suggesting a role for LPA in early mammalian development. In keratinocytes, and possibly other cell types, LPA-induced cell proliferation appears to be indirect as it involves the production of transforming growth factor in a PTX-dependent manner (6) . In confluent keratinocyte cultures, LPA stimulates terminal differentiation, while its topical application to mouse skin induces thickening of the epidermis (6) .

A somewhat puzzling finding is that in certain cell types, the mitogenic action of LPA can be reproduced by PA (but not other glycerophospholipids (6, 26) ), whereas PA does not appear to act on the LPA receptor. The dose-response relationships of PA and LPA largely overlap, implying that PA action is not mediated by LPA impurities in the PA used (26) . Whether this effect of PA is receptor-mediated is still obscure. Clearly, one major challenge is now to reconcile the mitogenic action of PA with that of LPA (see Ref. 2 for further discussion).

LPA, but not peptide growth factors, can mimic serum in inducing invasion of carcinoma and hepatoma cells into monolayers of mesothelial cells (28) . The underlying mechanism is not clear but may well involve both increased cell adhesion and enhanced cell motility. Other lipids, including PA, have little or no effect, but bacterial PLD does mimic LPA action (28) . The latter result is consistent with the finding that PLD treatment of cells may result in activation of endogenous LPA receptors.()

Like many mitogens, LPA induces rapid changes in the actin-based cytoskeleton. In serum-deprived fibroblasts, LPA stimulates focal adhesion assembly and stress fiber formation (29) . Assembly of new focal adhesions will lead to increased cell adhesion, while stress fiber formation may contribute to the build-up of contractile forces as occurs during wound repair. In neuroblastoma and PC12 cells, LPA triggers acute growth cone collapse and neurite withdrawal (9, 23) , a process driven by contraction of the cortical actin cytoskeleton rather than by loss of adhesion to the substratum (23, 30) . By stimulating neurite withdrawal, LPA tends to inhibit and reverse the morphological differentiation of neuroblastoma cells (2) . The in vivo significance of this intriguing phenomenon remains to be addressed. Given the abundance of the putative LPA receptor in brain (12) and the responsiveness of neuronal cells to LPA, it is tempting to speculate that LPA may serve a neuromodulator role, either under normal physiological conditions or during traumatic injury of the nervous system (discussed in Ref. 1). From a signal transduction point of view, the early cytoskeletal and late mitogenic effects of LPA are particularly intriguing since they cannot be explained by known second messenger pathways, as will be outlined below.


Structure-Activity Relationship

The structural features of LPA that are important for its biological activity are only beginning to be understood. A consistent finding is that maximal LPA activity is observed with long acyl chains (C to C) and that activity decreases with decreasing chain length; the shorter chain species, such as lauroyl- and decanoyl-LPA, show little or no activity (26, 31) . Human A431 carcinoma cells are particularly convenient for structure-activity analysis because of their high sensitivity to LPA in terms of Ca mobilization (EC for oleoyl-LPA, 0.2 nM(31) ). A recent study with these cells has revealed the importance of the free phosphate group; replacement of this moiety by either of three different phosphonates results in almost complete loss of activity; similarly, the methyl and ethyl esters of LPA are virtually inactive (31) .

Ether-linked LPA acts on the LPA receptor, albeit with less potency than the corresponding ester-linked LPA, at least in A431 cells (31) . In platelet aggregation assays, on the other hand, ether-linked LPAs appear to be the most potent species (32) . One possibility to explain this discrepancy is that platelets and carcinoma cells express different LPA receptors. It appears that the sn-1 as well as the sn-3 enantiomers of ether-linked LPA have about equal activity (31, 32) , with both isomers showing cross-desensitization. This apparent lack of stereospecificity is somewhat unexpected, since stereospecificity is often a critical feature of physiological receptor ligands. The fact that ether-linked LPAs behave as bona fide receptor agonists may be of physiological relevance; ether-linked LPAs are potential metabolites of 1-alkyl-2-acyl-glycerophospholipids (such as PAF), which are found in significant amounts in circulating cells such as neutrophils and macrophages. It will now be important to find out whether ether-linked LPA can occur extracellularly, either in physiological events or under pathological conditions.

Thus far, structure-activity studies have not identified specific LPA receptor antagonists (but see Ref. 33 for potential candidates). The only known LPA antagonist is suramin; this polyanionic compound abolishes both early and late responses to LPA in a reversible manner (23, 26), apparently by inhibiting LPA-receptor binding (12) .

In summary, although many features of the LPA structure are important for biological activity, the phosphate group is most critical. In a simple model (31) , the acyl chain may serve to anchor the LPA molecule in a hydrophobic cavity of the receptor in such a way that the glycerol-linked phosphate group is optimally oriented for direct interaction with positively charged residues in an extracellular region of the receptor. Such specific binding of the phosphate moiety would then trigger a conformational change that activates the receptor. Obviously, a direct test of this tentative model awaits cloning of the LPA receptor cDNA. It will then be possible to examine by site-directed mutagenesis how the LPA receptor detects and binds its ligand.


G Protein-mediated Signal Transduction

At least four G protein-mediated signaling pathways have been identified in the action of LPA. These are (i) stimulation of PLC and PLD, (ii) inhibition of adenylyl cyclase, (iii) activation of Ras and the downstream Raf/MAP kinase pathway, and (iv) tyrosine phosphorylation of focal adhesion proteins in concert with remodeling of the actin cytoskeleton in a Rho-dependent manner. Of these pathways, inhibition of adenylyl cyclase and stimulation of Ras signaling are sensitive to PTX. A simplified and tentative outline of the various signaling cascades is given in Fig. 2and will be further discussed below.


Figure 2: Proposed scheme of signaling pathways transduced by the LPA receptor. In this scheme, the LPA receptor couples to both PTX-sensitive and PTX-insensitive G proteins (G and G, respectively) to trigger various effector pathways. The free subunit of G directly inhibits adenylyl cyclase, while the dimers are thought to mediate LPA-induced Ras-GTP accumulation via an intermediary protein tyrosine kinase (inhibited by genistein and staurosporine). Active Ras then initiates the Raf/MAP kinase cascade to stimulate cell proliferation. The G protein is proposed to trigger PLC-mediated Rho signaling independently of classic phosphoinositide-derived second messengers. Perhaps, the PLC-induced loss of polyphosphoinositides may somehow provide the signal for Rho activation. Of note, it cannot be excluded that stimulation of PLC and Rho activation are parallel rather than sequential events; if so, PLC and Rho might be regulated by distinct G subunits (cf. G-mediated inhibition of adenylyl cyclase and activation of Ras). A putative protein tyrosine kinase (inhibited by tyrphostin) may act upstream of Rho. See text for further details and references. AC, adenylyl cyclase; IP, inositol trisphosphate; DG, diacylglycerol; PKC, protein kinase C.



Phospholipase Activation

When added at nanomolar doses, LPA activates phosphoinositide-specific PLC with consequent Ca mobilization and protein kinase C activation via a PTX-insensitive G protein (G family) in a great variety of cell types, ranging from Xenopus oocytes to mammalian neuronal cells (Refs. 1 and 2; see Ref. 31 for an update of LPA-responsive mammalian cell types). In fibroblasts, LPA also liberates arachidonic acid from prelabeled lipids (3) , but it has not yet been established whether this results from enhanced PLA activity or, more indirectly, from stimulation of a diacylglycerol lipase, or both. LPA also activates PLD to generate PA (and free choline) from phosphatidylcholine (34, 35) in a protein kinase C-dependent manner, as with many PLC-activating agonists (36) . The signaling function, if any, of newly generated PA is still obscure. It should be emphasized that, although enhanced phospholipid hydrolysis mediates diverse early cellular responses to LPA, it cannot account for LPA-induced cell proliferation (1, 2, 3) .

G-mediated Inhibition of Adenylyl Cyclase

In fibroblasts, LPA rapidly lowers cAMP levels following forced stimulation of adenylyl cyclase (3) . This effect is abolished by PTX and hence attributable to G-mediated inhibition of adenylyl cyclase. By this mechanism LPA may counteract the effects of cAMP-raising agonists, which are often growth inhibitory. It remains to be seen whether non-fibroblastic cell types respond similarly to LPA.

G-mediated Ras Signaling

LPA-induced fibroblast proliferation is almost completely inhibited by PTX and yet not attributable to known PTX-sensitive second messenger pathways. The critical G-mediated mitogenic event is activation of Ras (37) and its downstream effectors, particularly the Raf/MAP kinase cascade (38, 39, 40) . It seems that Ras is necessary for both the early, transient phase and the late, sustained phase of MAP kinase activation by LPA (38, 41) . Other G-coupled receptors, including the thrombin receptor (37) , also couple to rapid Ras activation, at least in a fibroblast context (reviewed in Refs. 1 and 2). It thus appears that G-coupled receptors and receptor protein tyrosine kinases utilize a similar mechanism for mitogenic signaling.

While the biochemical steps between tyrosine kinase receptors and Ras activation have been elucidated in much detail, little is still known about how G mediates Ras activation. A first clue comes from COS cell studies showing that rather than subunits of G are responsible for Ras-dependent MAP kinase activation (41, 42) . Furthermore, in Rat-1 cells expressing a inhibitor polypeptide, LPA-induced Ras signaling is significantly reduced (43) . dimers are thought to couple to an as yet unidentified effector that may stimulate, either directly or indirectly, GDP/GTP exchange on Ras, but it is equally well possible that acts on Ras via a GTPase-activating protein rather than a GDP/GTP exchanger. Whatever the precise mechanism, the G-Ras pathway has been proposed to comprise a non-receptor tyrosine kinase, since tyrosine kinase inhibitors (genistein and staurosporine) selectively inhibit LPA-induced (but not epidermal growth factor-induced) Ras-GTP accumulation (37, 40) . Unraveling the molecular details of the G-Ras connection is one of the major challenges for future studies.

Phosphatidylinositol 3-Kinase Activation

Many mitogens activate the p85/p110 phosphatidylinositol (PI) 3-kinase in their target cells and so does LPA. LPA-induced PI 3-kinase activity has been found in 3T3 cells (Ref. 44; but see ref. 45 for conflicting data) and in megakaryoblastic cells (46) . Whatever its precise link to the LPA receptor and physiological function, PI 3-kinase does not seem to be essential for mitogenic signaling, since microinjection of a neutralizing antibody does not affect LPA-induced DNA synthesis (47) .

Rho Signaling: Protein Tyrosine Phosphorylation and Cytoskeletal Changes

The least understood branch of the LPA receptor signaling cascade is the one that involves the Ras-related Rho protein. It accounts for LPA-induced cytoskeletal reorganizations, actomyosin-based force generation, and tyrosine phosphorylation of cytoskeleton-associated proteins. This pathway is somehow associated with PLC activation. In fibroblasts, G protein-linked receptor agonists that stimulate PLC also induce rapid tyrosine phosphorylation of focal adhesion proteins, and LPA is no exception to the rule (40, 44, 48) . The major tyrosine-phosphorylated substrates in LPA-treated fibroblasts comprise the 125-kDa ``focal adhesion kinase'' (FAK), paxillin, and a p130 protein (48) , originally identified as putative v-Src substrates. FAK is an integrin-linked tyrosine kinase that associates with the Src and Fyn tyrosine kinases, paxillin, and adaptor proteins and likely plays an important role in integrin-mediated signal transduction (49) . Immunofluorescence studies show that these LPA-induced tyrosine phosphorylations are accompanied by the recruitment of FAK, paxillin, talin, vinculin, as well as protein kinase C- to focal adhesion sites, resulting in rapid reassembly of focal adhesions and actin stress fibers (50, 51, 52) . Microinjected active Rho mimics exogenous LPA in recruiting tyrosine-phosphorylated proteins to focal adhesion sites, followed by stress fiber formation (29, 50) .

In other words, Rho-GTP links the LPA receptor to cytoskeletal events and protein tyrosine phosphorylations, but the details of the underlying signaling events remain enigmatic, as one is hampered by not knowing the biochemical targets of Rho. Current evidence indicates that active Rho-GTP directs the assembly of actin-based multimolecular complexes at the plasma membrane (such as focal adhesions; see Ref. 53 for review). Inactivation of endogenous Rho by the Botulinum C3 ADP-ribosyltransferase inhibits not only LPA-induced cytoskeletal effects but also tyrosine phosphorylation of FAK (44) . Tyrphostin 25, a tyrosine kinase inhibitor, inhibits the effects of LPA on the actin cytoskeleton but not the effects of microinjected Rho, suggesting that a non-receptor tyrosine kinase lies upstream of Rho (45) . Here is an interesting parallel with the mechanism proposed for LPA-induced Ras activation (37, 40) .

In the tentative model of Fig. 2, the PTX-insensitive G protein is proposed to mediate both PLC activation and Rho signaling. In the simplest model consistent with the fibroblast data, stimulation of PLC and Rho activation are sequential rather than parallel events. Although LPA-induced Rho signaling appears to be associated with PLC activation (independent of the Ras-MAP kinase cascade), it is independent of PLC-mediated Ca mobilization and/or protein kinase C activation (48, 50) . Perhaps the receptor-mediated decrease in phosphoinositide levels somehow provides the signal for Rho activation. Alternatively, phospholipid metabolites other than inositol phosphates and diacylglycerol might play a role.

A somewhat different picture holds for LPA-induced growth cone collapse and neurite retraction in neuroblastoma and PC12 cells. In these cells, Rho function appears essential for LPA-induced generation of contractile, actomyosin-based forces in the cortical cytoskeleton. Yet, there is no close correlation between PLC activation and Rho-dependent cytoskeletal changes, since phosphoinositide-hydrolyzing neuropeptides fail to evoke neurite withdrawal (23) . LPA-induced neurite retraction is accompanied by rapid activation of the p60 tyrosine kinase, but cause-effect relationships are not clear (23) . ADP-ribosylation of RhoA in growing neuroblastoma and PC12 cells causes extensive cell flattening (just opposite to what is observed in fibroblasts), probably as a result of reduced cytoskeletal tension, followed by prominent neurite outgrowth and growth arrest, very similar to what is observed after long term serum starvation (30) . These findings suggest that, in neuronal cells, Rho acts in a LPA signal transduction cascade that regulates actomyosin contractility in a Ca-independent manner and thereby directs neurite behavior; some basal Rho activity is probably necessary to maintain actomyosin-based tension and cell shape (30, 53). As a note of caution, the formal possibility remains that ADP-ribosylated Rho causes subtle modifications in the actomyosin system and thereby prevents it from proper functioning. Microinjection of active Rho-GTP should provide further answers.


Concluding Remarks

After having long been regarded as just a metabolic intermediate, LPA has now emerged as a multifunctional signaling molecule. Much has already been learned about LPA as a platelet-derived messenger, but numerous challenges remain. On the biological side, there are questions such as what are the precise biological functions of LPA and what cell types (other than platelets) produce and release LPA? It seems more than likely that platelet-derived LPA stimulates cell proliferation at sites of injury and inflammation, presumably in synergy with other platelet-derived factors, but this needs to be tested in vivo. At the molecular level, cDNA cloning and characterization of a functional G protein-linked receptor for LPA is of obvious importance; it will then be possible to address the tissue and cell type distribution of the LPA receptor and also whether there are multiple LPA receptor subtypes. Finally at the signal transduction level, there is the challenge of delineating the biochemical steps that couple the LPA receptor to activation of Ras and Rho. Ultimately, continued study of these questions should provide a detailed picture of the biological functions and mode of action of LPA as an intercellular messenger.

  
Table: Biological responses to LPA

For primary references see text and Refs. 1 and 2, unless indicated otherwise.



FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. This work was supported by the Dutch Cancer Society and the Netherlands Organization for Scientific Research.

§
To whom correspondence should be addressed. Tel.: 31-20-512-1971; Fax: 31-20-512-1989.

The abbreviations used are: LPA, lysophosphatidic acid; PLC, phospholipase C; PA, phosphatidic acid; PLA, phospholipase A; PLD, phospholipase D; PAF, platelet-activating factor; PTX, pertussis toxin; MAP, mitogen-activated protein; PI, phosphatidylinositol; FAK, focal adhesion kinase.

P. L. Hordijk and W. H. Moolenaar, unpublished observations.

C. Limatola and W. H. Moolenaar, unpublished observations.

Sphingosine 1-phosphate is structurally similar to LPA and exerts many LPA-like effects, including mitogenesis (19), PLC activation, stress fiber formation, and neurite retraction (K. Jalink and W. H. Moolenaar, unpublished observations); but cross-desensitization experiments indicate that sphingosine 1-phosphate does not act on the LPA receptor (31).

K. Jalink and W. H. Moolenaar, unpublished observations.


ACKNOWLEDGEMENTS

I thank my colleagues in the Netherlands Cancer Institute for helpful discussions. I have cited reviews when possible and apologize to those whose original work could not be cited because of space limitations.


REFERENCES
  1. Moolenaar, W. H.(1994) Trends Cell Biol. 4,213-219 [CrossRef]
  2. Jalink, K., Hordijk, P. L., and Moolenaar, W. H.(1994) Biochim. Biophys. Acta 1198, 185-196 [CrossRef][Medline] [Order article via Infotrieve]
  3. Van Corven, E. J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H.(1989) Cell 59, 45-54 [Medline] [Order article via Infotrieve]
  4. Tigyi, G., Dyer, D. L., and Miledi, R.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1908-1912 [Abstract]
  5. Tokumura, A., Iimori, M., Nishioka, Y., Kitahara, M., Sakashita, M., and Tanaka, S.(1994) Am. J. Physiol. 267, C204-C210
  6. Piazza, G. A., Ritter, J. L., and Baracka, C. A.(1995) Exp. Cell Res. 216, 51-64 [CrossRef][Medline] [Order article via Infotrieve]
  7. Kolodney, M. S., and Elson, E. L.(1993) J. Biol. Chem. 268, 23850-23855 [Abstract/Free Full Text]
  8. Zhang, Q., Checovich, W. J., Peters, D. M., Albrecht, R. M., and Mosher, D. F.(1994) J. Cell Biol. 127, 1447-1459 [Abstract]
  9. Tigyi, G., and Miledi, R.(1992) J. Biol. Chem. 267, 21360-21367 [Abstract/Free Full Text]
  10. Eichholtz, T., Jalink, K., Fahrenfort, I., and Moolenaar, W. H.(1993) Biochem. J. 291, 677-680 [Medline] [Order article via Infotrieve]
  11. Thomson, F. J., Perkins, L., Ahern, D., and Clark, M.(1994) Mol. Pharmacol. 45, 718-728 [Abstract]
  12. Van der Bend, R. L., Brunner, J., Jalink, K., van Corven, E. J., Moolenaar, W. H., and van Blitterswijk, W. J.(1992) EMBO J. 11, 2495-2501 [Abstract]
  13. Jalink, K., van Corven, E. J., and Moolenaar, W. H.(1990) J. Biol. Chem. 265, 12232-12239 [Abstract/Free Full Text]
  14. Gerrard, J. M., and Robinson, P.(1989) Biochim. Biophys. Acta 1001, 282-285 [Medline] [Order article via Infotrieve]
  15. Billah, M. M., Lapetina, E. G., and Cuatrecasas, P.(1981) J. Biol. Chem. 256, 5399-5403 [Free Full Text]
  16. Thompson, F. J., and Clark, M. A.(1995) Biochem. J. 306, 305-309 [Medline] [Order article via Infotrieve]
  17. Fukami, K., and Takenawa, T.(1992) J. Biol. Chem. 267, 10988-10993 [Abstract/Free Full Text]
  18. Thumser, A. E. A., Voysey, J. E., and Wilton, D. C.(1994) Biochem. J. 301, 801-806 [Medline] [Order article via Infotrieve]
  19. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S.(1991) J. Cell Biol. 114, 155-167 [Abstract]
  20. Van der Bend, R. L., de Widt, J., van Corven, E. J., Moolenaar, W. H., and van Blitterswijk, W. J.(1992) Biochim. Biophys. Acta 1125, 110-112 [Medline] [Order article via Infotrieve]
  21. Xie, M., and Low, M. G.(1994) Arch. Biochem. Biophys. 312, 254-259 [CrossRef][Medline] [Order article via Infotrieve]
  22. Thompson, F. J., and Clark, M. A.(1994) Biochem. J. 300, 457-461 [Medline] [Order article via Infotrieve]
  23. Jalink, K., Eichholtz, T., Postma, F. R., van Corven, E. J., and Moolenaar, W. H.(1993) Cell Growth & Differ. 4, 247-255
  24. Jalink, K., Moolenaar, W. H., and van Duijn, B.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1857-1861 [Abstract]
  25. Stoll, L. L., Denning, G. M., Kasner, N. A., and Hunninghake, G. W. (1994) J. Biol. Chem. 269, 4254-4259 [Abstract/Free Full Text]
  26. Van Corven, E. J., van Rijswijk, A., Jalink, K., van der Bend, R. L., van Blitterswijk, W. J., and Moolenaar, W. H.(1992) Biochem. J. 281, 163-169 [Medline] [Order article via Infotrieve]
  27. Kobayashi, T., Yamano, S., Murayama, S., Ishikawa, H., and Tokumura, A. (1994) FEBS Lett. 351, 38-40 [CrossRef][Medline] [Order article via Infotrieve]
  28. Imamura, F., Horai, T., Mukai, M., Shinkai, K., Sawada, M., and Akedo, H.(1993) Biochem. Biophys. Res. Commun. 193, 497-503 [CrossRef][Medline] [Order article via Infotrieve]
  29. Ridley, A. J., and Hall, A.(1992) Cell 70, 389-399 [Medline] [Order article via Infotrieve]
  30. Jalink, K., van Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H.(1994) J. Cell Biol. 126, 801-810 [Abstract]
  31. Jalink, K., Hengeveld, T., Mulder, S., Postma, F. R., Simon, M.-F., Chap, H., van der Marel, G. A., van Boom, J. H., van Blitterswijk, W. J., and Moolenaar, W. H.(1995) Biochem. J. 307, 609-616 [Medline] [Order article via Infotrieve]
  32. Simon, M. F., Chap, H., and Douste-Blazy, L.(1982) Biochem. Biophys. Res. Commun. 108, 1743-1750 [Medline] [Order article via Infotrieve]
  33. Sugiura, T., Tokumura, A., Gregory, L., Nouchi, T., Weintraub, S. T., and Hanahan, D. J.(1994) Arch. Biochem. Biophys. 311, 358-368 [CrossRef][Medline] [Order article via Infotrieve]
  34. Van der Bend, R. L., de Widt, J., van Corven, E. J., Moolenaar, W. H., and van Blitterswijk, W. J.(1992) Biochem. J. 285, 235-240 [Medline] [Order article via Infotrieve]
  35. Ha, K.-S., Yeo, E.-J., and Exton, J. H.(1994) Biochem. J. 302, 55-59
  36. Exton, J. H.(1994) Biochim. Biophys. Acta 1212, 26-42 [Medline] [Order article via Infotrieve]
  37. Van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., and Moolenaar, W. H.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1257-1261 [Abstract]
  38. Cook, S. J., Rubinfield, B., Albert, I., and McCormick, F.(1993) EMBO J. 12, 3475-3485 [Abstract]
  39. Howe, L. R., and Marshall, C. J.(1993) J. Biol. Chem. 268, 20717-20720 [Abstract/Free Full Text]
  40. Hordijk, P. L., Verlaan, I., van Corven, E. J., and Moolenaar, W. H. (1994) J. Biol. Chem. 269, 645-651 [Abstract/Free Full Text]
  41. Cook, S., and McCormick, F.(1994) Nature 369, 361-362 [Medline] [Order article via Infotrieve]
  42. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S.(1994) Nature 369, 418-420 [CrossRef][Medline] [Order article via Infotrieve]
  43. Koch, W. J., Hawes, B. E., Allen, L. F., and Lefkowitz, R. J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12706-12710 [Abstract/Free Full Text]
  44. Kumagai, N., Morii, N., Fujisawa, K., Nemoto, Y., and Narumiya, S. (1993) J. Biol. Chem. 268, 24535-24538 [Abstract/Free Full Text]
  45. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A.(1995) J. Cell Sci. 108, 225-233 [Abstract/Free Full Text]
  46. Vemuri, G. S., and Rittenhouse, S. E.(1994) Biochem. Biophys. Res. Commun. 202, 1619-1623 [CrossRef][Medline] [Order article via Infotrieve]
  47. Roche, S., Koegl, M., and Courtneidge, S. A.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9185-9189 [Abstract]
  48. Seufferlein, T., and Rozengurt, E.(1994) J. Biol. Chem. 269, 9345-9351 [Abstract/Free Full Text]
  49. Schaller, M. D., and Parsons, J. T.(1994) Curr. Opin. Cell Biol. 6, 705-710 [Medline] [Order article via Infotrieve]
  50. Ridley, A. J., and Hall, A.(1994) EMBO J. 13, 2600-2610 [Abstract]
  51. Barry, S. T., and Critchley, D. R.(1994) J. Cell Sci. 107, 2033-2045 [Abstract/Free Full Text]
  52. Chrzanowska-Wodnicka, M., and Burridge, K.(1994) J. Cell Sci. 107, 3643-3654 [Abstract/Free Full Text]
  53. Hall, A.(1994) Annu. Rev. Cell Biol. 10, 31-54 [CrossRef]
  54. Hii, C. S. T., Oh, S. Y., Schmidt, S. A., Clark, K. J., and Murray, A. W.(1994) Biochem. J. 303, 475-479 [Medline] [Order article via Infotrieve]
  55. Shiono, S., Kawamoto, K., Yoshida, N., Kondo, T., and Inagami, T. (1993) Biochem. Biophys. Res Commun. 193, 667-663 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.