David J. Fischer

Lysophosphatidic Acid‐Induced Neurite Retraction in PC12 Cells: Control by Phosphoinositide‐Ca2+ Signaling and Rho

Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP‐ribosylation of the small‐molecular‐weight G protein Rho by the Clostridium botulinum C‐3 toxin. LPA induced a transient rise in the level of inositol 1,4,5‐trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)‐Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long‐term phorbol ester treatment—all diminished neurite retraction by interfering with LPA‐induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15‐min treatment with 4β‐phorbol 12‐myristate 13‐acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP‐Ca2+ second messenger system appears necessary for the Rho‐mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA‐induced morphoregulatory response.

Identification of Edg1 receptor residues that recognize sphingosine 1-Phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg120 and Arg292 ion pair with the phosphate, whereas the acidic Glu121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35S]GTPγS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Lysophosphatidic acid-induced neurite retraction in PC12 cells: neurite-protective effects of cyclic AMP signaling.

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric Gi proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl‐cyclic AMP prevented neurite retraction. The neurite‐protective effect of forskolin was blocked by Rp‐adenosine 3′,5′‐phosphorothioate. Forskolin and dibutyryl‐cyclic AMP both failed to protect neurites in A126‐1B2 and 123.7 cells, which lack cyclic AMP‐activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP‐activated protein kinase‐dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP‐ribosylation of Rho by the Clostridium botulinum C‐3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA‐induced neurite retraction. LPA activates Gq‐ and Gi‐linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor‐activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.

The Faciogenital Dysplasia Gene Product FGD1 Functions as a Cdc42Hs-specific Guanine-Nucleotide Exchange Factor

The Rho family of small GTP-binding proteins plays important roles in the regulation of actin cytoskeleton organization and cell growth. Activation of these GTPases involves the replacement of bound GDP with GTP, a process catalyzed by the Dbl-like guanine-nucleotide exchange factors, all of which seem to share a putative catalytic motif termed the Dbl homology (DH) domain, followed by a pleckstrin homology (PH) domain. Here we have examined the role of a Dbl-like molecule, the faciogenital dysplasia gene product (FGD1), which when mutated in its Dbl homology domain, cosegregates with the developmental disease Aarskog-Scott syndrome. We report that a polypeptide of FGD1 encompassing the DH and PH domains can bind specifically to the Rho family GTPase Cdc42Hs and stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. Microinjection of this FGD1 polypeptide into Swiss 3T3 fibroblast cells induces the formation of peripheral actin microspikes, similar to that previously observed when cells were injected with a constitutively active form of Cdc42Hs. This effect of FGD1 on actin organization is readily inhibited by coinjection of a dominant-negative mutant of Cdc42Hs. Examination of NIH 3T3 cells expressing the FGD1 fragment revealed that similar to cells expressing Dbl, two independent elements downstream of Cdc42Hs, the Jun NH2-terminal kinase and the p70 S6 kinase, became activated. Hence, our results indicate that FGD1, through its DH and PH domains, acts as a Cdc42Hs-specific guanine-nucleotide exchange factor and suggest that the Cdc42Hs GTPase may have a role in mammalian development.

Molecular basis for lysophosphatidic acid receptor antagonist selectivity

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.

Identification of a Novel Growth Factor-like Lipid, 1-O-cis-Alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (Alkenyl-GP) That Is Present in Commercial Sphingolipid Preparations

Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 μmwas only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065–C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.

Pharmacological Characterization of Phospholipid Growth-Factor Receptors

Abstract: The phospholipid growth‐factor (PLGF) terminology is proposed to describe a group of endogenous glycerol‐ and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus‐coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl‐glycerophosphate, cyclic‐phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA‐activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT‐PCR results reinforce the coexpression of PSP24α and different members of the EDG1‐7 receptors in the same cell. Stable heterologous expression of the PSP24α, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose‐response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.

Structural Features of EDG1 Receptor‐Ligand Complexes Revealed by Computational Modeling and Mutagenesis

ABBY L. PARRILL,a,b DANIEL L. BAKER,c DE-AN WANG,c DAVID J. FISCHER,c DEBRA L. BAUTISTA,b JAMES VAN BROCKLYN,d SARAH SPIEGEL,d AND GABOR TIGYIc bDepartment of Chemistry, University of Memphis, Memphis, Tennessee 38152, USA cDepartment of Physiology, University of Tennessee, Memphis, Tennessee 38163, USA dDepartment of Biochemistry and Molecular Biology, Georgetown University, Washington, District of Columbia 20007, USA

Dynamic modeling of EDG1 receptor structural changes induced by site-directed mutations

Abstract EDG1 is a member of the G protein coupled receptor family and serves as a cellular receptor responsive to phospholipids. EDG1 binds sphingosine-1-phosphate (SPP) with high affinity and lysophophatidic acid (LPA) with low affinity. A model has been developed, based on an experimentally based model of the structure of rhodopsin, to evaluate the features that contribute to the different binding affinities of phospholipids for EDG1. The residues predicted by the model to be important in binding have previously been reported. Twenty mutations expressed transiently in HEK293 cells were evaluated by radioligand binding assays and MAP-kinase assays of receptor activation. Seventeen of these mutations are well explained by the current model. The remaining three mutations and three additional control mutations have been modeled using molecular dynamics. Changes in the exposed surface areas of amino acids in the binding pocket reflect the changes in SPP binding observed for the modeled mutations.

Characterization of Endogenous and Heterologously Expressed LPA Receptor Subtypes

Lysophosphatidic acid (LPA), alkenyl-glycerophosphate (alkenyl-GP), and cyclicphosphatidic acid (cyclic-PA) are naturally occurring phospholipid growth factors (PLGFs). PLGFs elicit diverse biological effects via the activation of G-proteincoupled receptors in a variety of cell types. The cellular and signaling properties of the LPA analogues were compared in NIH3T3 cells,1 where both LPA and alkenylGP induced cell proliferation, decreased cAMP levels, and activated the ERK and JNK MAP kinases. In contrast, cyclic-PA inhibited cell proliferation, increased cAMP levels, and slightly inhibited the ERK and JNK MAP kinases. All three PLGFs dose-dependently induced Ca2+ transients and induced the formation of stress fibers. Since the three LPA analogues elicited different signaling and cellular responses, heterologous desensitization patterns were established, by monitoring changes in intracellular Ca2+, to determine if the three analogues activated the same or different receptors. When NIH3T3 cells were desensitized to LPA, they were no longer responsive to either alkenyl-GP or cyclic-PA. Cells that were desensitized to alkenylGP were no longer responsive to cyclic-PA and only partially responsive to LPA, whereas cells that were desensitized to cyclic-PA were only partially responsive to both alkenyl-GP and LPA. These patterns demonstrated that subtypes of PLGF receptors are simultaneously expressed in the same cell, distinguishing between alkenyl-GP and cyclic-PA, but are all activated by LPA. We propose a model to describe three pharmacologically distinct receptor subtypes in NIH3T3 cells (FIG. 1). Type I receptors are selectively activated by LPA, type II receptors are activated by both LPA and alkenyl-GP, while type III receptors are selectively activated by cyclic-PA, as well as LPA and alkenyl-GP. The responsiveness of the cells, when measuring Ca2+ transients, to the LPA analogues was dependent upon the length of serum starvation. Cells that had not been serum-starved were unresponsive to all three LPA analogues. The cells first became responsive to both LPA and alkenyl-GP 2 hours after the removal of serum, while the maximum responses were not achieved until 12 hours. In comparison, the cells did not become responsive to the application of cyclic-PA until 12 hours after