Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors
Introduction
Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are important lipid mediators generated from membrane phospholipids upon cell activation. Besides acting as intracellular second messengers [1], [2], they have been recognized only recently as extracellular activators of a family of G protein-coupled receptors (GPCRs) [3], [4]. The S1P binding GPCRs so far comprise EDG1, EDG3, EDG5, EDG6 and EDG8. High-affinity receptors for LPA include EDG2, EDG4 and EDG7 (for review, see [5], [6], [7]). Additional GPCRs responding to lipid-like mediators have been identified recently: TDAG8 responding to psychosin [8], GPR68 (=OGR1, ovarian cancer GPCR1) activated by sphingosylphosphorylcholine (SPC) [9], G2A stimulated by lysophosphatidylcholine (LPC) [10] and GPR4 responding to SPC and LPC [11]. The large variety of biologically active lipids and the recent pairings of such lipids with orphan GPCRs imply that several additional, not yet identified, orphan lipid GPCRs may exist. In addition, the effects of the lipid mediators S1P and LPA cannot be fully explained by the current repertoire of S1P and LPA binding receptors.
As lysophospholipid mediators in general are implicated in many biological processes such as angiogenesis [12], cardiac development [13], platelet aggregation [14] and induction/suppression of apoptosis [15], emphasis should be given to (i) identification of new lipid-binding GPCRs and (ii) identification of additional GPCRs as potential targets for the modulation by S1P and/or LPA.
Human gpr3, gpr6 and gpr12 have been cloned by several independent groups [16], [17], [18] as members of the GPCR superfamily exhibiting high similarity to each other. Devoid of a natural ligand, they are currently classified as orphan GPCRs. Among receptors with known ligands, cannabinoid (CB), endothelial differentiation gene (EDG) and melanocortin (MC) GPCRs are the closest relatives, although not so closely related as to predict identical ligands or physiological function. Northern blot and RT-PCR analysis have shown the receptors to be expressed predominantly in cerebral tissues [17], [19], [20], [21], [22], suggesting a ligand with neurotransmitter or neuromodulator function. Expression of human gpr3 (originally named hACCA=human adenylate cyclase constitutive activator) in various mammalian cell lines conferred constitutive signalling via Gαs type of G proteins and generation of cyclic adenosine monophosphate (cAMP) onto these cells [23]. However, it remained open whether stimulation of adenylate cyclase (AC) was the result of “true” constitutive activity (CA) or the presence of a ubiquitous ligand in the culture medium. In addition, it is not known whether gpr6 and gpr12 share constitutive activation of the cAMP signalling cascade with gpr3, thus constituting a family of constitutively active GPCRs.
In this study, we confirm CA of gpr3 and extend these findings onto the closely related receptors gpr6 and gpr12. In addition, evidence will be presented that all three receptors not only exhibit constitutive signalling towards the Gαs pathway, but also to the Gαi/o signalling cascade, thus constituting a family with dual coupling characteristics. Various functional assays (cAMP second messenger assays, mobilization of intracellular Ca2+, agonist-induced internalization) were applied to confirm S1P and DHS1P as activators of the gpr3, gpr6 and gpr12 family. In addition, the complete rat gpr3 receptor has been cloned, showing similar functional behaviour to that of its human counterpart.
Section snippets
Materials
S1P, LPA, LPC, SPC, Sph, suramin, fatty acid free BSA (FAF-BSA), PTX, TG, forskolin (fsk) and isobutylmethylxanthine (IBMX) were purchased from Sigma (Deisenhofen, Germany), and DHS1P and the lipid library from Biomol Research Laboratories (Plymouth Meeting, USA). Fluo-4 and pluronic acid were taken from Molecular Probes (MoBiTec; Göttingen, Germany), the mammalian expression vector pcDNA3.1 from Invitrogen (Carlsbad, USA), the FuGENE6 transfection reagent from Roche (Basel, Swizerland), the
Similarity of gpr3, gpr6 and gpr12 to classes of lipid GPCRs
Phylogenetic analysis (Fig. 1A) showed that gpr3, gpr6 and gpr12 (gpr3 human: accession no. L32831; gpr6 human: accession no. L36150; gpr12 human: accession no. U18548) represent a subfamily of receptors. Their closest relatives with identified functions consist of the CB, MC and EDG receptor family. gpr3, gpr6 and gpr12 display 42% similarity on the amino acid level to CB (activated by anandamide), 43% similarity to EDG (main ligands S1P and LPA) and 44% similarity to MC receptors.
Discussion
The results of the present study identify the GPCRs gpr3, gpr6 and gpr12 [16], [17], [18] as a family of constitutively active receptors with the ability to regulate positively the AC signalling cascade in the absence of an activating ligand (Fig. 2). The lipid mediators S1P and DHS1P have been identified as modulators of this GPCR family in functional assays and internalization studies. In addition, rat gpr3 (Fig. 1C) has been cloned based on the availability of a 334-bp fragment referred to
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