Edg-8 receptors are preferentially expressed in oligodendrocyte lineage cells of the rat CNS.

The messenger RNA for endothelial differentiation gene 8 receptors is known to be expressed almost exclusively in the rat CNS, but the nature of the expressing cells has not been defined. Using an antibody specific for endothelial differentiation gene 8, we investigated the immunohistochemical localization of endothelial differentiation gene 8 receptors in the rat CNS. Immunopositive staining was detected in a subset of glial cells distributed throughout the brain and spinal cord, including both gray and white matter, but not in the dorsal root ganglion. The distribution and morphological similarity in comparative immunostaining for endothelial differentiation gene 8 and various glial markers suggested that endothelial differentiation gene 8 is preferentially expressed in NG2-positive oligodendrocyte progenitor cells in adult rat brains. Counts of endothelial differentiation gene 8-positive cells and NG2-positive cells in the forebrain revealed that a subset of NG2-positive cells was endothelial differentiation gene 8-positive, and that the ratio of endothelial differentiation gene 8-positive cells to NG2-positive cells varied from region to region. In 17-day-old embryonic brains, the endothelial differentiation gene 8 distribution was similar to that of an oligodendrocytic marker, 2',3'-cyclic nucleotide 3'-phosphodiesterase. These data suggest that endothelial differentiation gene 8 receptors are preferentially expressed in oligodendrocyte lineage cells including oligodendrocyte progenitor cells and immature/maturating oligodendrocytes in rat CNS, and that they might have important functions in oligodendrocytic maturation and myelination.

Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells.

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor.

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.

Molecular cloning and characterization of another leukotriene B4 receptor.

Leukotriene B(4) is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B(4), for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B(4), named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B(4) induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B(4) in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B(4) receptor will eventually lead to a better understanding of the classification of leukotriene B(4) receptors and reconsideration of the pathophysiological role of leukotriene B(4).

Transformation of BALB3T3 cells caused by over-expression of rat CD98 heavy chain (HC) requires its association with light chain: mis-sense mutation in a cysteine residue of CD98HC eliminates its transforming activity.

CD98 is a 125-kDa glycoprotein (GP125) consisting of an 85-kDa heavy chain (HC) and a 40-kDa light chain (LC), and is highly expressed on the cell surface of activated lymphocytes and various tumor cells. In addition to the regulatory role of CD98HC in L-, y(+)L- and Xc-amino-acid transport systems, which are principally mediated by CD98LC, we have reported transforming activity of human CD98HC. In this study, we established and analyzed BALB3T3 clones transfected with cDNAs encoding wild-type and mutated rat CD98HC proteins designated as BrH/Wild, C103S, C325S and 103/325, in which 103 and/or 325 cysteine were intact or replaced with serine. Flow cytometry with anti-rat CD98HC MAb B3 revealed that wild-type and mutated CD98HC transfectants expressed almost the same amounts of rat CD98HC proteins on the cell surface. Immunoprecipitation with B3 revealed that exogenous rat CD98HC proteins were associated with endogenous mouse CD98LC by a disulfide bond in BrH/Wild and C325S, but not in C103S and 103/325 transfectants. These transfectants showed similar doubling times and leucine and arginine transport activities, as compared with BALB3T3 and control transfectants in monolayer culture. Wild-type and C325S transfectants, however, formed much larger anchorage-independent colonies than C103S, 103/325 and control transfectants in soft agar. In addition, wild-type and C325S transfectants showed tumorigenicity in nude mice, although C103S, 103/325 and control transfectants did not. These findings indicate that over-expression of CD98HC and its disulfide-linkage with CD98LC at the cell surface result in malignant transformation of murine fibroblasts.

The molecular characterization and tissue distribution of the human cysteinyl leukotriene CysLT(2) receptor.

Cysteinyl leukotrienes (CysLTs), slow-reacting substances of anaphylaxis, are lipid mediators known to possess potent proinflammatory action. Pharmacological studies using CysLTs indicate that at least two classes of G protein-coupled receptors (GPCRs), named CysLT(1) and CysLT(2), exist; the former is sensitive and the latter is resistant to the CysLT(1) antagonists currently used to treat asthma. Although the CysLT(1) receptor gene has been recently cloned, the molecular identity of the CysLT(2) receptor has remained elusive. Here we show that the pharmacological profile of an orphan GPCR (PSEC0146) is consistent with that of the CysLT(2) receptor. In human embryonic kidney 293 cells that express the PSEC0146 cDNA, leukotriene C(4) (LTC(4)) and leukotriene D(4) (LTD(4)) induce equal increases in intracellular calcium mobilization; these increases are not affected by CysLT(1) antagonists. Additionally, [(3)H]LTC(4) specifically binds to membranes from COS-1 cells transiently transfected with PSEC0146. Large amounts of the PSEC0146 mRNA are found in human heart, placenta, spleen, and peripheral blood leukocytes but not in the lung and the trachea. Pharmacological feature and expression studies will eventually lead to a better understanding of the classification of CysLT receptors, possibly leading to a reconsideration of the pathological and physiological role of CysLTs.

An evolutionarily conserved G-protein coupled receptor family, SREB, expressed in the central nervous system.

We report here a novel family of G-protein coupled receptor (GPCR) which is extraordinarily conserved among vertebrate species. This family, designated SREB (Super Conserved Receptor Expressed in Brain), consists of at least three members, termed SREB1, SREB2, and SREB3. SREB members share 52-63% amino acid identity with each other and show relatively high similarity to previously known amine amine GPCRs (approximately 25% identity). Amino acid sequence identity between human and rat orthologues is 97% for SREB1 and 99% for SREB3, while the SREB2 sequence is surprisingly completely identical between the species. Furthermore, amino acid sequence of zebrafish SREB2 and SREB3 are 94 and 78% identical to mammal orthologues. Northern blot analysis revealed that SREB members are predominantly expressed in the brain regions and genital organs. Radiation hybrid analysis localized SREB1, SREB2, and SREB3 genes to different human chromosomes, namely 3p21-p14, 7q31 and Xp11, respectively. The high sequence conservation and abundant expression in the central nervous system suggest the existence of undiscovered fundamental neuronal systems consisting of SREB family members and their endogenous ligand(s).

The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors.

A cDNA encoding a novel G-protein coupled receptor (GPCR) was isolated from a human cerebral cortex cDNA library by low stringency hybridization screening. This putative seven-transmembrane domain receptor of 469 amino acids was designated SALPR (Somatostatin- and Angiotensin- Like Peptide Receptor). SALPR shares the highest amount of amino acid similarity with the somatostatin (35% with SSTR5) and angiotensin receptors (31% with AT1). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the SALPR mRNA is predominantly expressed in human brain regions, particularly the substantia nigra and pituitary, although the mRNA can also be detected in the peripheral tissues, albeit at low levels. Chromosomal mapping by radiation hybrid analysis localized the human SALPR gene to the chromosome 5p15.1-5p14. Transient expression of SALPR in COS-1 cells did not produce any binding sites for somatostatin or angiotensin II, indicating the necessity for further study to discover its ligand and physiological significance.