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 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.
Uhlenbrock
Kirsten
K
Aventis Pharma Germany, Disease Group Cardiovascular, Industriepark Höchst, Frankfurt/Main, Germany. kirsten.uhlenbrock@aventis.com
Gassenhuber
Hans
H
Kostenis
Evi
E
D000262:Adenylyl Cyclases / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins; D008246:Lysophospholipids; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology
C097350:GPR12 protein, human; C092794:GPR3 protein, human; C091672:GPR6 protein, human; D008024:Ligands; D008164:Luminescent Proteins; D008246:Lysophospholipids; D008563:Membrane Lipids; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C060504:dihydrosphingosine 1-phosphate; C060506:sphingosine 1-phosphate; D013498:Suramin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D013110:Sphingosine