Uhlenbrock Kirsten, Gassenhuber Hans, Kostenis Evi
Aventis Pharma Germany, Disease Group Cardiovascular, Industriepark Höchst, Frankfurt/Main, Germany.
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+) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.
到目前为止，已克隆并鉴定了五种针对溶血磷脂鞘氨醇-1-磷酸（S1P）的G蛋白偶联受体（GPCR）。我们在此报告gpr3、gpr6和gpr12作为S1P-GPCR家族新成员的鉴定情况。当在HEK293细胞中瞬时表达时，gpr3、gpr6和gpr12可导致腺苷酸环化酶（AC）的组成性激活，其幅度与完全激活的G(α)(s)偶联受体相似。在含有活性炭处理血清（无脂质）的培养基中培养转染细胞可显著降低环磷酸腺苷（cAMP）水平，提示存在类脂质配体。将一个包含200种生物活性脂质的文库应用于记录细胞内Ca(2+)动员的功能测定中。S1P和二氢鞘氨醇-1-磷酸（DHS1P）被鉴定为具有纳摩尔级EC(50)值的功能性激活剂。在S1P和溶血磷脂酸（LPA）受体拮抗剂苏拉明存在的情况下，gpr3、gpr6和gpr12介导的通过S1P的细胞内Ca(2+)动员增强。除了组成性激活G(α)(s)型G蛋白外，这三种受体都能够组成性激活抑制性G(α)(i/o)蛋白：（i）在百日咳毒素存在的情况下，gpr3、gpr6和gpr12介导的AC刺激增强；（ii）G(α)(i)的过表达显著降低对细胞内cAMP水平的刺激作用。使用gpr6绿色荧光蛋白（GFP）融合蛋白可在完整的HEK293细胞中观察到激动剂（S1P）介导的内化。总之，我们的数据表明gpr3、gpr6和gpr12是一类组成性激活的受体，可与G(α)(s)和G(α)(i)型G蛋白双重偶联。AC的组成性激活和Ca(2+)的动员可被鞘磷脂S1P和DHS1P调节，这为S1P受体家族增加了三个新成员。
