Bektas Meryem, Barak Larry S, Jolly Puneet S, Liu Hong, Lynch Kevin R, Lacana Emanuela, Suhr Ki-Beom, Milstien Sheldon, Spiegel Sarah
Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, USA.
Biochemistry. 2003 Oct 28;42(42):12181-91. doi: 10.1021/bi035051y.
The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.
溶血磷脂、溶血磷脂酸、1-磷酸鞘氨醇和鞘氨醇磷酸胆碱（SPC）是调节多种生物过程的生物活性脂质分子。尽管溶血磷脂酸和1-磷酸鞘氨醇的特异性G蛋白偶联受体已得到充分表征，但对SPC受体的了解却少得多。据报道，卵巢癌G蛋白偶联受体1（OGR1）是SPC的高亲和力受体，其密切相关的同源物GPR4是SPC的高亲和力受体，对溶血磷脂酰胆碱（LPC）的亲和力较低。然而，在一项检测配体结合特异性的功能试验中，我们发现SPC、LPC或其他相关溶血磷脂均未诱导GPR4从质膜内化。同样，这些溶血脂质也未在表达GPR4的细胞中诱导β-抑制蛋白2-GFP从胞质溶胶向质膜的转位。然而，当这些细胞与G蛋白偶联受体激酶2共转染时，在未添加配体的情况下，β-抑制蛋白2-GFP聚集在细胞质小泡中，这让人联想到通常在激动剂刺激GPCR后观察到的小泡标记。此外，SPC和LPC均未刺激GTPγS与从表达GPR4的细胞制备的膜结合，也未激活ERK1/2。令人惊讶的是，GPR4的强制表达抑制了由多种刺激诱导的ERK1/2激活，包括SPC、1-磷酸鞘氨醇，甚至表皮生长因子（EGF）。总的来说，我们的结果表明SPC和LPC不是GPR4的配体，并且该受体可能组成性地抑制ERK1/2激活。
