Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling.

In peripheral nerves, Schwann cells (SCs) form contacts with axons, other SCs, and extracellular matrix components that are critical for their migration, differentiation, and response to injury. Here, we report that lysophosphatidic acid (LPA), an extracellular signaling phospholipid, regulates the morphology and adhesion of cultured SCs. Treatment with LPA induces f-actin rearrangements resulting in a "wreath"-like structure, with actin loops bundled peripherally by short orthogonal filaments. The latter appear to anchor the SC to a laminin substrate, because they colocalize with the focal adhesion proteins, paxillin and vinculin. SCs also respond to LPA treatment by forming extensive cell-cell junctions containing N-cadherin, resulting in cell clustering. Pharmacological blocking experiments indicate that LPA-induced actin rearrangements and focal adhesion assembly involve Rho pathway activation via a pertussis toxin-insensitive G-protein. The transcript encoding LP(A1), the canonical G-protein-coupled receptor for LPA, is upregulated after sciatic nerve transection, and SCs cultured from lp(A1)-null mice exhibit greatly diminished morphological responses to LPA. Cultured SCs can release an LPA-like factor implicating SCs as a potential source of endogenous, signaling LPA. These data, together with the previous demonstration of LPA-mediated SC survival, implicate endogenous receptor-mediated LPA signaling in the control of SC development and function.

Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3.

Sphingosine 1-phosphate (S1P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S1P receptors have been identified in mammals: LP(B1)/EDG-1, LP(B2)/H218/AGR16/EDG-5, LP(B3)/EDG-3, LP(B4)/NRG-1/EDG-8, and LP(C1)/EDG-6. One of these receptors, LP(B1), has recently been shown to be essential for mouse embryonic development. Here we disrupted the lp(B3) gene in mice, resulting in the complete absence of lp(B3) gene, transcript, and LP(B3) protein. LP(B3)-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LP(B3) deletion on S1P-induced signal transduction pathways. Wild-type MEF cells expressed lp(B1), lp(B2), and lp(B3) but neither lp(B4) nor lp(C1), and they were highly responsive to S1P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LP(B3)-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LP(B3) receptor in LP(B3)-null MEF cells restored S1P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LP(B3) in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor.

Expression and function of lysophosphatidic acid receptors in cultured rodent microglial cells.

Microglia are the resident tissue macrophages of the central nervous system. They are rapidly activated by a variety of insults; and recently, receptors linked to cytoplasmic Ca(2+) signals have been implicated in such events. One potential class of receptors are those recognizing lysophosphatidic acid (LPA). LPA is a phospholipid signaling molecule that has been shown to cause multiple cellular responses, including increases in cytoplasmic calcium. We examined whether any of the known LPA receptor genes (lp(A1)/Edg2, lp(A2)/Edg4, and lp(A3)/Edg7) are expressed by cultured mouse or rat microglia. Reverse transcriptase-polymerase chain reaction indicated that mouse microglia predominantly expressed the lp(A1) gene, whereas rat microglia predominantly expressed lp(A3). Although LPA induced increases in the cytoplasmic Ca(2+) concentration in both microglial preparations, the responses differed substantially. The Ca(2+) signal in rat microglia occurred primarily through Ca(2+) influx via the plasma membrane, whereas the Ca(2+) signal in mouse microglia was due to release from intracellular stores. Only at high concentrations was an additional influx component recruited. Additionally, LPA induced increased metabolic activity in mouse (but not rat) microglial cells. Our findings provide evidence for functional LPA receptors on microglia. Thus, LPA might play an important role as a mediator of microglial activation in response to central nervous system injury.

The mouse lp(A3)/Edg7 lysophosphatidic acid receptor gene: genomic structure, chromosomal localization, and expression pattern.

The extracellular signaling molecule, lysophosphatidic acid (LPA), mediates proliferative and morphological effects on cells and has been proposed to be involved in several biological processes including neuronal development, wound healing, and cancer progression. Three mammalian G protein-coupled receptors, encoded by genes designated lp (lysophospholipid) receptor or edg (endothelial differentiation gene), mediate the effects of LPA, activating similar (e.g. Ca(2+) release) as well as distinct (neurite retraction) responses. To understand the evolution and function of LPA receptor genes, we characterized lp(A3)/Edg7 in mouse and human and compared the expression pattern with the other two known LPA receptor genes (lp(A1)/Edg2 and lp(A2)/Edg4non-mutant). We found mouse and human lp(A3) to have nearly identical three-exon genomic structures, with introns upstream of the coding region for transmembrane domain (TMD) I and within the coding region for TMD VI. This structure is similar to lp(A1) and lp(A2), indicating a common ancestral gene with two introns. We localized mouse lp(A3) to distal Chromosome 3 near the varitint waddler (Va) gene, in a region syntenic with the human lp(A3) chromosomal location (1p22.3-31.1). We found highest expression levels of each of the three LPA receptor genes in adult mouse testes, relatively high expression levels of lp(A2) and lp(A3) in kidney, and moderate expression of lp(A2) and lp(A3) in lung. All lp(A) transcripts were expressed during brain development, with lp(A1) and lp(A2) transcripts expressed during the embryonic neurogenic period, and lp(A3) transcript during the early postnatal period. Our results indicate both overlapping as well as distinct functions of lp(A1), lp(A2), and lp(A3).

Lysophospholipid receptors.

Lysophospholipids (LPs), including lysophosphatidic acid and sphingosine 1-phosphate, produce many cellular effects. However, the prolonged absence of any cloned and identified LP receptor has left open the question of how these lipids actually bring about these effects. The cloning and functional identification of the first LP receptor, lp(A1)/vzg-1, has led rapidly to the identification and classification of multiple orphan receptors/expression sequence tags known by many names (e.g. edg, mrec1.3, gpcr26, H218, AGR16, nrg-1) as members of a common cognate G protein-coupled receptor family. We review features of the LP receptor family, including molecular characteristics, genomics, signaling properties, and gene expression. A major question for which only partial answers are available concerns the biological significance of receptor-mediated LP signaling. Recent studies that demonstrate the role of receptor-mediated LP signaling in the nervous system, cardiovascular system, and other organ systems indicate the importance of this signaling in development, function, and pathophysiology and portend an exciting time ahead for this growing field.

Lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) is a simple bioactive phospholipid with diverse physiological actions on many cell types. LPA induces proliferative and/or morphological effects and has been proposed to be involved in biologically important processes including neurogenesis, myelination, angiogenesis, wound healing, and cancer progression. LPA acts through specific G protein-coupled, seven-transmembrane domain receptors. To date, three mammalian cognate receptor genes, lp(A1)/vzg-1/Edg2, lp(A2)/Edg4, and lp(A3)/Edg7, have been identified that encode high-affinity LPA receptors. Here, we review current knowledge on these LPA receptors, including their isolation, function, expression pattern, gene structure, chromosomal location, and possible physiological or pathological roles.

Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior.

Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, lp(A1)/vzg-1/edg-2, was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of lp(A1) results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among lp(A1)((-/-)) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and/or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an lp receptor gene and indicate that LPA signaling through the LP(A1) receptor is required for normal development of an inborn, neonatal behavior.

Functional comparisons of the lysophosphatidic acid receptors, LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7 in neuronal cell lines using a retrovirus expression system.

Lysophosphatidic acid (LPA) is a potent lipid mediator with diverse physiological actions on a wide variety of cells and tissues. Three cognate G-protein-coupled receptors have been identified as mammalian LPA receptors: LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7. The mouse forms of these genes were analyzed in rodent cell lines derived from nervous system cells that can express these receptors functionally. An efficient retrovirus expression system was used, and each receptor was heterologously expressed in B103 rat neuroblastoma cells that neither express these receptors nor respond to LPA in all assays tested. Comparative analyses of signaling pathways that are activated within minutes of ligand delivery were carried out. LPA induced cell rounding in LP(A1)- and LP(A2)-expressing cells. By contrast, LP(A3) expression resulted in neurite elongation in B103 cells and inhibited LPA-dependent cell rounding in TR mouse neuroblast cells that endogenously express LP(A1) and LP(A2) but not LP(A3). Each of the receptors could couple to multiple G-proteins and induced LPA-dependent inositol phosphate production, mitogen-activated protein kinase activation, and arachidonic acid release while inhibiting forskolin-induced cAMP accumulation, although the efficacy and potency of LPA varied from receptor to receptor. These results indicate both shared and distinct functions among the three mammalian LPA receptors. The retroviruses developed in this study should provide tools for addressing these functions in vivo.

Neurobiology of receptor-mediated lysophospholipid signaling. From the first lysophospholipid receptor to roles in nervous system function and development.

Identification of the first lysophospholipid receptor, LPA1/Vzg-1, cloned by way of neurobiological analyses on the embryonic cerebral cortex, has led to the realization and demonstration that there exist multiple, homologous LP receptors, including those encoded by a number of orphan receptor genes known as "Edg," all of which are members of the G-protein-coupled receptor (GPCR) superfamily. These receptors interact with apparent high affinity for lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P or SPP), and are referred to based upon their functional identity as lysophospholipid receptors: LPA and LPB receptors, respectively, with the expectation that additional subgroups will be identified (i.e., LPC, etc.). Here an update is provided on insights gained from analyses of these receptor genes as they relate to the nervous system, particularly the cerebral cortex, and myelinating cells (oligodendrocytes and Schwann cells).

Genomic characterization of the lysophosphatidic acid receptor gene, lp(A2)/Edg4, and identification of a frameshift mutation in a previously characterized cDNA.

To understand the regulation, evolution, and genetics of lp(A2)/Edg4, a second lysophosphatidic acid receptor gene, we characterized its complete cDNA sequence, genomic structure, and chromosomal location. The full-length mouse transcript sequence was determined using rapid amplification of cDNA ends. Southern blot and restriction fragment length polymorphism segregation analyses revealed that the mouse gene was present as a single copy and located at the middle of Chromosome 8 near the mutations for myodystrophy (myd) and "kidney-anemia-testes" (kat). This region is syntenic with human chromosome 19p12, where the human genomic clone containing the lp(A2) gene (EDG4) was mapped. Sequence analysis of genomic clones demonstrated that both mouse and human transcripts were encoded by three exons, with an intron separating the coding region for transmembrane domain VI. Reverse transcriptase-PCR demonstrated that the three exons were spliced in all mouse tissues shown to express the transcript. Finally, in a comparison of all human lp(A2) sequences present in the database, we identified several sequence variants in multiple tumors. One such variant (a G deletion) in the initially characterized Edg4 cDNA clone (derived from an ovarian tumor) results in a frameshift mutation near the 3' end of the coding region. In addition to increasing our understanding of the mechanisms underlying lysophosphatidic acid signaling and lysophospholipid receptor gene evolution, these results have important implications regarding the genomic targeting and oncogenic potential of lp(A2).

A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs).

A missing component in the experimental analysis of cell signaling by extracellular lysophospholipids such as lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) has been cloned receptors. Through studies on the developing brain, the first such receptor gene (referred to as vzg-1) was identified, representing a member of the G-protein coupled receptor (GPCR) super family (1). Here we review the neurobiological approach that led to both its cloning and identification as a receptor for LPA, along with related expression data. Summarized sequence and genomic structure analyses indicate that this first, functionally identified receptor is encoded by a member of a growing gene family that divides into at least two subgroups: genes most homologous to the high-affinity LPA receptor encoded by vzg-1, and those more homologous to an orphan receptor gene edg-1 that has recently been identified as a S1P receptor. A provisional nomenclature is proposed, based on published functional ligand actions, amino acid composition and genomic structure whereby the receptors encoded by these genes are referred to as lysophospholipid (LP) receptors, with subgroups distinguished by letter and number subscripts (e.g., LPA1 for Vzg-1, and LPB1 for Edg-1). Presented expression data support the recently published work indicating that members of the LPB1 subgroup are receptors for the structurally-related molecule, S1P. The availability of cloned LP receptors will enhance the analysis of the many documented LP effects, while their prominent expression in the nervous system indicates significant but as yet unknown roles in development, normal function, and neuropathology.

Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate.

The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lpA1 (also referred to as vzg-1 or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lpB1, lpB2, and lpB3) were isolated, corresponding to human/rat Edg-1, rat H218/AGR16, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44-52% identical), the three lpB genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lpA1 and lpA2. Unlike lpA1 and lpA2, which contain multiple coding exons, all lpB members contained a single coding exon. Heterologous expression of individual lpB members in a hepatoma cell line (RH7777), followed by 35S-GTPgammaS incorporation assays demonstrated that each of the three LPB receptors conferred sphingosine-1-phosphate-dependent, but not lysophosphatidic acid-dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple sphingosine-1-phosphate receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family.

Complete cDNA sequence, genomic structure, and chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26.

The lpA1/Gpcr26 locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3' rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5' sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor, Edg1, but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 in Mus spretus, and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for the vzg-1/lpA1 subfamily of receptors compared to those of homologous orphan receptor genes.

Structure and expression of an inhibitor of fungal polygalacturonases from tomato.

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.