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).

Lysophosphatidic acid receptor gene vzg-1/lpA1/edg-2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain.

The growth-factor-like phospholipid lysophosphatidic acid (LPA) mediates a wide variety of biological functions. We recently reported the cloning of the first G-protein-coupled receptor for LPA, called ventricular zone gene-1 (vzg-1/lpA1/edg-2) because its embryonic central nervous system (CNS) expression is restricted to the neocortical ventricular zone (Hecht et al. [1996] J. Cell Biol. 135:1071-1083). Vzg-1 neural expression diminishes at the end of the cortical neurogenetic period, just before birth. Here, we have investigated the subsequent reappearance of vzg-1 expression in the postnatal murine brain, by using in situ hybridization and northern blot analyses. Vzg-1 expression was undetectable by in situ hybridization at birth, but reappeared in the hindbrain during the 1st postnatal week. Subsequently, expression expanded from caudal to rostral, with peak expression observed around postnatal day 18. At all postnatal ages, vzg-1 expression was concentrated in and around developing white matter tracts, and its expansion, peak, and subsequent downregulation closely paralleled the progress of myelination. Double-label in situ hybridization studies demonstrated that vzg-1-expressing cells co-expressed mRNA encoding proteolipid protein (PLP), a mature oligodendrocyte marker, but not glial fibrillary acidic protein (GFAP), an astrocyte marker. Consistent with this, vzg-1 mRNA expression was reduced by 40% in the brains of jimpy mice, which exhibit aberrant oligodendrocyte differentiation and cell death. Together with our characterization of vzg-1 during cortical neurogenesis, these data suggest distinct pre- and postnatal roles for LPA in the development of neurons and oligodendrocytes and implicate lysophospholipid signaling as a potential regulator of myelination.

Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex.

Neocortical neuroblast cell lines were used to clone G-protein-coupled receptor (GPCR) genes to study signaling mechanisms regulating cortical neurogenesis. One putative GPCR gene displayed an in situ expression pattern enriched in cortical neurogenic regions and was therefore named ventricular zone gene-1 (vzg-1). The vzg-1 cDNA hybridized to a 3.8-kb mRNA transcript and encoded a protein with a predicted molecular mass of 41-42 kD, confirmed by Western blot analysis. To assess its function, vzg-1 was overexpressed in a cell line from which it was cloned, inducing serum-dependent "cell rounding." Lysophosphatidic acid (LPA), a bioactive lipid present in high concentrations in serum, reproduced the effect seen with serum alone. Morphological responses to other related phospholipids or to thrombin, another agent that induces cell rounding through a GPCR, were not observed in vzg-1 overexpressing cells. Vzg-1 overexpression decreased the EC50 of both cell rounding and Gi activation in response to LPA. Pertussis toxin treatment inhibited vzg-1-dependent LPA-mediated Gi activation, but had no effect on cell rounding. Membrane binding studies indicated that vzg-1 overexpression increased specific LPA binding. These analyses identify the vzg-1 gene product as a receptor for LPA, suggesting the operation of LPA signaling mechanisms in cortical neurogenesis. Vzg-1 therefore provides a link between extracellular LPA and the activation of LPA-mediated signaling pathways through a single receptor and will allow new investigations into LPA signaling both in neural and nonneural systems.

GATA-binding proteins regulate the human gonadotropin alpha-subunit gene in the placenta and pituitary gland.

The human glycoprotein hormone alpha-subunit gene is expressed in two quite dissimilar tissues, the placenta and anterior pituitary. Tissue-specific expression is determined by combinations of elements, some of which are common and others of which are specific to each tissue. In the placenta, a composite enhancer confers specific expression. It contains four protein-binding sites: two cyclic AMP (cAMP) response elements that bind CREB, a trophoblast-specific element that binds TSEB, and a sequence motif, AGATAA, that matches the consensus binding site for a family of transcription factors termed the GATA-binding proteins. In pituitary gonadotropes, the cAMP response elements remain important for expression, TSEB is absent, and elements further upstream participate in tissue-specific expression. Here we establish a regulatory role for the GATA element in both the placenta and pituitary by demonstrating that a mutation of this element decreases alpha-subunit gene expression 15-fold in JEG-3 human placental cells and 2.5-fold in alpha T3-1 mouse pituitary gonadotropes. In JEG-3 cells, human GATA-2 (hGATA-2) and hGATA-3 are highly expressed and both proteins bind to the alpha-subunit gene GATA element. In alpha T3-1 cells, the GATA motif is bound by mouse GATA-2 (mGATA-2) and an mGATA-4-related protein. Cotransfection of hGATA-2 or hGATA-3 into alpha T3-1 cells activates the alpha-subunit gene threefold. These studies establish a role for the GATA-binding proteins in placental and pituitary alpha-subunit gene expression, significantly expanding the known target genes of GATA-2, GATA-3, and perhaps GATA-4.

Coordinate control of the alpha- and beta-subunit genes of human chorionic gonadotropin by trophoblast-specific element-binding protein.

The alpha- and beta-subunit genes of hCG are coordinately regulated in the trophectoderm of the early embryo and placenta. Placenta-specific expression of the alpha-subunit gene is determined by a composite enhancer made of three clustered components: cAMP-responsive elements, a GATA site, and the trophoblast-specific element (TSE). We have investigated the basis of placenta-specific expression of the major hCG beta-subunit gene, hCG beta 5. Enhancement of expression localizes to the region from -305 to -279, whereas full cAMP regulation requires the region from -305 to -249. Four DNAse-I footprints are present, three of which can be competed by the TSE element from the alpha-subunit gene. Methylation interference establishes that binding to the element located in the key region for expression, from -301 to -275, requires contacts with a CCNNNGGG core sequence that matches the alpha-subunit gene TSE. Sequence-specific DNA affinity chromatography using the alpha-subunit gene TSE allows purification of TSE-binding protein. This purified protein binds specifically to the key element, -301 to -275, and to at least two additional TSE elements clustered in the regulatory region of the hCG beta 5 gene. We conclude that both the alpha- and beta-subunit genes of hCG require the placenta-specific factor TSE-binding protein for expression, providing a mechanism for their coordinate regulation in placental cells.

A method of identifying and isolating a unique member of a multigene family: application to a trypanosome surface antigen gene.

A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.