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

Abstract

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.

Molecular Cloning and Characterization of a Novel Human G-protein-coupled Receptor, EDG7, for Lysophosphatidic Acid

“…Thus, EDG2 seems to recognize LPAs with both saturated and unsaturated fatty acids like EDG4. It has been previously demonstrated that LPAs with unsaturated fatty acids evoked greater Ca 2ϩ responses of the human epidermoid carcinoma cell line A431 than LPAs with saturated fatty acids (30). In addition, Tokumura et al (31) reported that degree of unsaturation in the acyl moiety of LPA affects proliferation of cultured vascular smooth muscle cells from rat aorta by LPA.…”

Lysophosphatidic Acid-induced Mitogenesis Is Regulated by Lipid Phosphate Phosphatases and Is Edg-receptor Independent

“…Ours is not the first report of activity of phosphonate compounds in assays of LPA receptor activation. In 1991, Proll and Clark reported that an LPA analog with a phosphonate head group inhibits cAMP accumulation (50), but Moolenaar's group later showed that the same compound had little activity with respect to calcium mobilization (12). We have synthesized and tested a similar compound in a GTP␥S binding assay at each Edg/LPA receptor and found that it is an agonist at Edg-2 but has very little activity at Edg-4 or Edg-7.…”

“…Hopper et al (56) reported calcium mobilization in response to LPA with an EC 50 of 5 nM in MDA MB-231, and Moolenaar's group (12) has reported an LPA EC 50 of 0.2 nM in A-431 cells, measuring calcium mobilization in a buffer system containing 1 mM calcium. The potency of LPA with respect to inhibition of cAMP accumulation has been measured and reported in two previous papers by us to be 20 nM (13) and 52 nM (14), and these experiments were performed in 1.8 mM calcium.…”

“…In a wide variety of tissue culture cells, LPA elicits Ca 2ϩ mobilization with an EC 50 of 0.2-10 nM (11,12), inhibition of cAMP accumulation with an EC 50 of 50 -100 nM (1,13,14), cell rounding (15), and other cytoskeletal rearrangements such as formation of stress fibers and focal adhesions (16), fibrinogen binding (17), and neurite retraction (18) also with nanomolar potency.…”

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes

“…It is well known that LPA induces calcium mobilization [23,24]. As shown in Figure 5, LPAstimulated H # O # release was inhibited when cells were incubated with the intracellular calcium-chelator BAPTA-AM.…”