No abstract

Abstract. 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 LPAinduced 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.

Some agonists of G protein-coupled receptors, such as thrombin and lysophosphatidic acid (LPA), can promote cell proliferation via a pertussis toxin (PTX)-sensitive signaling pathway. While these agonists stimulate phospholipase C and inhibit adenylate cyclase, it appears that other, as-yet-unidentified, effector pathways are required for mitogenesis. Here we report that LPA and a thrombin receptor agonist peptide rapidly activate the protooncogene product p2l1 in quiescent fibroblasts. This activation is inhibited by PTX and yet not attributable to known PTX-sensitive G protein pathways, including stimulation of phospholipases, inhibition of adenylate cyclase, or modulation of ion channels. LPA-and peptide-induced p21w activation is inhibited by the tyrosine kinase inhibitor genistein, at doses that do not affect epidermal growth factor-induced p2l1 activation. Thus, a heterotrimeric G protein of the GI subfamily regulates activation of p21 by LPA and thrombin, possibly through an intermediary tyrosine kinase. This pathway may critically participate in mitogenic signaling downstream from certain G proteincoupled receptors.The signaling mechanisms by which growth factors stimulate cell proliferation have not been fully identified. Besides the widely studied ligands of receptor protein tyrosine kinases, certain agonists of G protein-coupled receptors also are capable of stimulating DNA synthesis in responsive cells. Examples of the latter class of mitogens include the protease thrombin (1, 2) and the lipid lysophosphatidic acid (LPA) (3-5). These otherwise unrelated agonists stimulate DNA synthesis in quiescent fibroblasts through a pertussis toxin (PTX)-sensitive signaling pathway (1-3, 5, 6). This assigns a central role for a heterotrimeric G protein of the G, subfamily in thrombin-and LPA-induced mitogenesis; however, the nature of the G1-mediated signal(s) is currently a matter of speculation.Thrombin cleaves and thereby activates its cognate seventransmembrane-domain receptor (7) to trigger G proteindependent stimulation of phospholipase C and PTIX-sensitive inhibition of adenylate cyclase (1, 2, 7-10); a synthetic peptide corresponding to the new N terminus of the cleaved receptor can serve as a full agonist of the cloned thrombin receptor (7-10). LPA, a bioactive phospholipid (5) produced and released by activated platelets (T. Eichholtz and W.H.M., unpublished results), appears to bind to its own G protein-coupled receptor to activate effector systems similar to thrombin (3-6, 11, 12). Yet it appears that activation ofthe phospholipase C-protein kinase C pathway is neither required nor sufficient for LPA-and thrombin-induced DNA synthesis, while it is doubtful whether PTX-sensitive inhibition of adenylate cyclase provides a bona fide mitogenic signal (for review see refs. 2 and 6). This implies that alternative G protein-effector routes must exist to account for mitogenesis.A major signaling event induced by ligands of receptor tyrosine kinases, such as epidermal growth factor (EGF) or insulin, is...

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with growth-factor-like activities [van Corven, Groenink, Jalink, Eichholtz & Moolenaar (1989) Cell 45, 45-54]. We have examined various structural analogues of LPA for their ability to stimulate DNA synthesis in quiescent fibroblasts. When the acyl-chain length is varied, the rank order of mitogenic potency is: 1-oleoyl LPA congruent to 1-palmitoyl LPA greater than 1-myristoyl LPA greater than 1-lauroyl LPA greater than 1-decanoyl LPA; the last compound shows almost no activity over the concentration range tested (1-100 microM). An ether-linked LPA (1-O-hexadecylglycerol 3-phosphate) has much decreased mitogenic activity as compared with the ester-linked analogue at concentrations less than 25 microM, and becomes cytotoxic at higher concentrations. Hexadecylphosphate, which lacks a glycerol backbone, has negligible activity. On a molar basis, diacyl phosphatidic acid (PA) is about equally potent as the corresponding LPA analogue, showing similar acyl-chain-length dependence; the data argue against the possibility that the mitogenic action of PA is due to contaminating traces of LPA. Although the short-chain analogues of LPA and PA fail to antagonize the action of long-chain (L)PAs, the polyanionic drug suramin inhibits LPA- and PA-induced, DNA synthesis in a reversible and dose-dependent manner, at concentrations [IC50 (concn. giving 50% inhibition) approximately 70 microM] that do not affect epidermal-growth-factor-induced DNA synthesis. Suramin appears to act in the early G0/G1 phase of the cell cycle, blocking immediate responses to LPA such as phosphoinositide hydrolysis. We conclude that both LPA and PA can function as growth-promoting phospholipids, with the fatty acid chain length being a major determinant of mitogenic potency.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormoneand growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoafTinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32p_ labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 -10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suraniin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.