Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors

Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated Galpha(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca2+ mobilization. SIP and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC50 values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca2+ Mobilization via S1P is enhanced. Besides constitutive activation of Gets type of G proteins, all three receptors are capable of constitutively activating inhibitory Galpha(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of Galpha(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to Galpha(s) and Galpha(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca2+](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors. (C) 2002 Elsevier Science Inc. All rights reserved.