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

Cell Signal. 2002 Nov;14(11):941-53. doi: 10.1016/s0898-6568(02)00041-4.


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 G(alpha)(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 Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(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 G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology
  • Cells, Cultured
  • Cyclic AMP / genetics
  • Cyclic AMP / metabolism
  • Eukaryotic Cells / metabolism*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / isolation & purification*
  • Green Fluorescent Proteins
  • Humans
  • Ligands
  • Luminescent Proteins
  • Lysophospholipids*
  • Membrane Lipids / metabolism*
  • Membrane Proteins / drug effects
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Receptors, Cell Surface / drug effects
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Receptors, G-Protein-Coupled*
  • Recombinant Fusion Proteins / pharmacology
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Sphingosine / analogs & derivatives*
  • Sphingosine / metabolism*
  • Sphingosine / pharmacology
  • Suramin / pharmacology


  • GPR12 protein, human
  • GPR3 protein, human
  • GPR6 protein, human
  • Ligands
  • Luminescent Proteins
  • Lysophospholipids
  • Membrane Lipids
  • Membrane Proteins
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • dihydrosphingosine 1-phosphate
  • sphingosine 1-phosphate
  • Suramin
  • Cyclic AMP
  • GTP-Binding Proteins
  • Adenylyl Cyclases
  • Sphingosine