Comparison of Intrinsic Activities of the Putative Sphingosine 1-Phosphate Receptor Subtypes to Regulate Several Signaling Pathways in Their cDNA-transfected Chinese Hamster Ovary Cells

Abstract

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [ 3 H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca 2؉ concentration ([Ca 2؉ ] i ) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca 2؉ ] i increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca 2؉ responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca 2؉ system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.Sphingosine 1-phosphate (S1P), 1 one of the sphingolipid metabolites, has recently been suggested to affect a variety of cellular processes (1, 2). These cellular responses elicited by S1P have first been ascribed to the intracellular action of the lipid, because S1P accumulated in the cells in response to some kinds of cytokines, and moreover, S1P induced Ca 2ϩ mobilization in a cell-free system (3-5). On the other hand, these S1P-induced responses are also accompanied by the stimulation of several early signaling events that are usually regulated by cell-surface receptors. These signaling events include activation of PLC (6 -9), an increase in [Ca 2ϩ ] i (10 -12), regulation of adenylyl cyclase (6, 9, 10, 13), and Rho activation (14, 15). The presence of the latter mechanism has been supported by the recent identification of several cDNAs encoding G-protein-coupled receptors for S1P, i.e. Edg-1, AGR16/H218, and .The transfection experiments of these S1P receptor subtypes demonstrated that these putative S1P receptors can actually couple to multiple signaling pathways. Thus, the previous transfection experiments suggest the involvement of these putative S1P receptor subtypes in the regulation of multiple signal...

Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3

“…The K d and maximal binding activity were estimated as 12.1 nM and 1.70 pmol\mg protein, respectively, based on Scatchard analysis ( Figure 5A, inset). The K d value was very similar to that for S1P-receptors expressed in CHO cells [27]. The binding affinity of Figure 6 Northern-blot analysis of putative S1P receptors Total RNA (10 µg) prepared from HAECs was electrophoresed to analyse the expression of Edg-1, Edg-3 and AGR16 (Edg-5) mRNA.…”

“…The migration of endothelial cells was quantified using a blind Boyden chamber apparatus using procedures essentially the same as those described in [27], except that endothelial cells and RPMI 1640 medium were used instead of CHO cells and Dulbecco's modified Eagle's medium. The number of cells that had migrated during 4 h to the lower surface was determined by counting the cells in four places under microscopy at i400 magnification (4HPF).…”

“…S1P receptor binding was performed essentially as described in [27]. The cells were incubated with [$H]S1P, from 3.1-50 nM, for dose-dependent binding.…”

“…Other experimental procedures have been described previously [27]. Specific S1P binding to its receptor was estimated by subtracting the radioactivity in the presence of 10 µM unlabelled S1P for Scatchard analysis and 1 µM unlabelled S1P for the competition experiments.…”

“…Furthermore, the lipid actions may be mediated through the putative S1P receptors, Edg-1 and Edg-3. [27]. The sources of all other reagents were the same as described previously [27][28][29][30].…”