Comparative analysis of human and rat S1P5 (edg8): differential expression profiles and sensitivities to antagonists
Introduction
The lysolipid phosphate mediators, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have attracted increasing attention as modulators of a variety of important biological functions [1], [2], [3], [4] and their list of biological activities is continuously growing.
S1P has been implicated in cell proliferation, modulation of cell motility [5], [6], induction/suppression of apoptosis [7], [8], in vitro and in vivo angiogenesis [9], tumor invasiveness [10], [11], platelet activation [12], and neurite retraction [13]. Cellular signaling by S1P involves activation of PLCβ and subsequent intracellular Ca2+ release [14], [15], activation of MAP-kinases [16], activation of inward rectifying K+-channels [17], [18], and inhibition and/or activation of adenylyl cyclase [14].
Both LPA and S1P are recognized to signal cells through a set of G protein-coupled receptors (GPCRs), formerly known as endothelial differentiation gene (edg) receptors. This family of GPCRs currently comprises eight members and, on the basis of their activating ligand, can be classified into two major groups: S1P1–5 [S1P1 (edg1), S1P2 (edg5), S1P3 (edg3), S1P4 (edg6), S1P5 (edg8)] are stimulated by S1P [19], [20], [21], [22], [23], LPA1–3 [LPA1 (edg2), LPA2 (edg4), LPA3 (edg7)] preferentially interact with LPA [24], [25].
Assignment of specific biological functions to certain receptor subtypes is hampered by: (1) the overlapping expression of S1P/LPA receptors [26], [27]; (2) activation of multiple, and in part redundant, signal transduction pathways [14], [15], [16], [26]; (3) incomplete selectivity of their activating ligands [28], [29]; and (4) poorly developed medicinal chemistry as specific antagonists for dissecting the pharmacology of individual subtypes are not yet available.
An important step to shed more light on the biological roles of the individual receptor subtypes would be to identify the complete set of receptors that respond to S1P and LPA and then to characterize the specific signaling and pharmacological properties.
Rat S1P5 (rS1P5) has recently been identified as the fifth S1P-responsive GPCR. It was originally cloned from rat pheochromocytoma cells, as a nerve growth factor regulated GPCR (nrg-1) [30] exhibiting greatest similarity to the family of S1P1–4 receptors. rS1P5 is expressed in spleen and adult brain white matter [23]. Malek et al.[28] demonstrated that rS1P5 is coupled to G proteins of the Gi/o- and G12-class, and inhibits activation of extracellular regulated kinase (ERK) in CHO cells. Much less is known about the human orthologue of rS1P5. Im et al.[31] recently published preliminary expression and functional studies of human S1P5 (hS1P5). hS1P5 is expressed in brain and peripheral tissues as determined by Northern blot analysis (multiple tissue dot blot); however, dot blot analysis revealed neither the transcript size nor the existence of tissue specific splice variants. In addition, low-level signals were detected in many tissues, making it hard to distinguish specific expression from background. Given the differential expression patterns of hS1P5 and rodent S1P5 receptors, we reasoned that additional pharmacological/biochemical differences may exist between the species homologues.
The present study was set out to address the following questions: (i) is hS1P5 an ubiquitously or specifically expressed gene, (ii) how many splice variants exist for hS1P5, and how are their tissue expression pattern, (iii) are the different expression patterns of hS1P5 and rS1P5 reflected in different sensitivities to agonists or antagonists, and (iv) does hS1P5 resemble its rat counterpart in the antiproliferative activity upon expression in mammalian cell lines?
Section snippets
Sources of materials
S1P, dhS1P, suramin, and fatty acid-free BSA were from Sigma. The lipid library was from Biomol Research Laboratories, Inc. NF023 was ordered from Tocris Cookson, CHO-K1 cells were obtained from the American Type Culture Collection, cell culture media and sera from Gibco BRL, the calcium fluorescent dye Fluo4 and pluronic acid from Molecular Devices, rat Northern blot membrane from Origene, human Northern blot membrane and the GC-melt PCR kit from Clontech. The expression vector PSPT18, the
Expression of hS1P5 and rS1P5
Im et al.[31] recently showed, via dot blot analysis, that hS1P5 is widespread in peripheral tissues and brain regions. To get more detailed information about the transcript size and putative splice variants, we examined the expression pattern of hS1P5 by Northern blot analysis (Fig. 1A). A prominent band migrating at 5.5 kilobases (kb) was seen in skeletal muscle, heart, and kidney, while lower abundance of RNA was observed in liver and placenta; no signal was detected in brain, thymus, spleen,
Discussion
Recently, hS1P5 has been discovered as the fifth member of the S1P receptor family [31]. Whereas many S1P receptors are ubiquitously expressed [27], [37], [38], hS1P5 displays a more specific expression pattern. Northern blot analysis of this study has revealed two new and interesting aspects of its expression: (1) hS1P5 is mainly expressed in the cardiovascular system, as opposed to rS1P5, that is exclusively expressed in brain, spleen, and skin; (2) hS1P5 exists as two splice variants with
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