Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1-phosphate-dependent increases in [35 S]guanosine 5-(3-O-thio)triphosphate binding with an EC 50 of 90 nM. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC 50 0.7 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G i/o ␣ proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G q/i ␣ protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K D ‫؍‬ 2 nM) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G i/o ␣ proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.Sphingosine 1-phosphate (S1P) 1 is a potent, extracellular lysolipid phosphoric acid mediator that is released, for example, during platelet activation (1). S1P elicits a wide variety of responses by cells; prominent among these are cell proliferation (2-4) and anti-apoptosis (5, 6) as well as a wide variety of other effects. S1P and the structurally related lysolipid mediator, lysophosphatidic acid (LPA), are recognized now to signal cells through a set of G protein coupled receptors known colloquially as the "Edg" receptors. Discovered initially as "orphan" receptors (7, 8), three members of the group, Edg-1, Edg-5, and Edg-3, have been shown to be S1P receptors. For example, Edg-1 mediates S1P activation of mitogen-activated protein kinase and inhibition of adenylyl cyclase in a pertussis toxindependent manner (9, 10). S1P activation of Edg-3 results calcium mobilization in a pertussis toxin-independent manner (11), while others found that this receptor coupled also, for example, to inhibition of adenylyl cyclase via G i/o ␣ protein (12). Likewise, several groups have shown that a third S1P receptor, Edg-5, couples also to G q/11 ␣ proteins (13, 14). All three S1P receptors signal in Xenopus oocytes, although Edg-1 signaling was dependent on co-injection with a chimeric G q ␣/G i ␣ protein (15). These data suggest that the three known S1P receptors interact with different signa...

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, ⑀. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABA A receptor ␥3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth ␥ subunit. The ⑀ subunit coassembles with GABA A receptor ␣ and ␤ subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. ␣1␤1⑀ GABA A receptors, like ␣1␤1␥2s receptors, are modulated by pentobarbital and the steroid 5␣-pregnan-3␣-ol-20-one but, unlike ␣1␤1␥2s receptors, are insensitive to flunitrazepam. Additionally, ␣1␤1⑀ receptors exhibit rapid desensitization kinetics, as compared with ␣1␤1 or ␣1␤1␥2s. Northern analysis demonstrates widespread expression of a large ⑀ subunit transcript in a variety of nonneuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the ⑀ subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

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43Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions 44 such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure 52demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This 53 study lays the groundwork for development of a diagnostic test that can be used as an aid for 54 distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric 55 illnesses with overlapping symptoms.

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