Functional analysis of a novel receptor for lysophosphatidic acid (LPA)

用LPA 1或LPA 4（也称为p2 γ 9/GPR 23）的表达载体稳定转染不能对溶血磷脂酸（LPA）应答的B103大鼠神经母细胞瘤细胞。我发现，在对LPA的反应中，LPA 4介导了B103-LPA 4细胞中的Ca^2+反应。此外，LPA 4显示激活Rho小GT3，导致包括细胞变圆和细胞聚集的形态学变化。虽然B103-LPA 1细胞在LPA刺激后也变圆，但它们的细胞变圆程度显著低于B103-LPA 4细胞。至于细胞聚集，LPA 1活化不诱导任何形态学变化。我观察到，与LPA 1不同，LPA 4不与百日咳毒素敏感的Gi/o蛋白偶联，这可能是LPA 1和LPA 4之间这些功能差异的原因。由于LPA 1在神经系统中起着关键作用，如脑的形成和神经病理性疼痛，我的数据提出了LPA 4在体内也可能具有神经元功能的可能性。实际上，在Western分析中，在富含LPA 4 mRNA的小鼠胚胎12天的脑中，通过抗血清成功地检测到LPA 4蛋白，并且在表达LPA 4的B103细胞中通过流式细胞术分析成功地检测到LPA 4蛋白。将携带适当同源重组事件而没有随机整合靶向载体的ES细胞的三个克隆注射到胚泡中。来自三个克隆之一的嵌合体导致靶向等位基因的种系传递，这通过Southern分析证实。

B103 rat neuroblastoma cells, which are unable to respond to lysophosphatidic acid (LPA), were stably transfected, with the expression vector for either LPA1 or LPA4 (also known as p2y9/GPR23). I found that, in response to LPA, LPA4 mediated Ca^<2+> responses in B103-LPA4 cells. Furthermore, LPA4 was shown to activate Rho small GTPase, resulting in the morphological changes including cell rounding and cell aggregation. Although B103-LPA1 cells also became rounded upon LPA stimulation, their degree of cell rounding was significantly less than that of B103-LPA4 cells. As for cell aggregation, LPA1 activation did not induce any morphological changes. My observation that, unlike LPA1, LPA4 did not couple to pertussis toxin-sensitive Gi/o proteins may account for these functional differences between LPA1 and LPA4. Because LPA1 plays critical roles in the nervous system such as brain formation and neuropathic pain, my data raise the possibility that LPA4 also may have neuronal functions in vivo.A rabbit polyclonal antiserum was raised against a synthetic peptide derived from mouse LPA4. Actually, LPA4 protein was successfully detected by the antiserum in Western analysis of mouse brain at embryonic day 12, which was rich in LPA4 mRNA, and flow cytometry analysis of the LPA4-expressing B103 cells.The LPA4 gene was disrupted in marine embryonic stem (ES) cells by homologous recombination. Three clones of ES cells carrying the proper homologous recombination events without random integration of the targeting vector were injected into blastocysts. Chimeras from one of the three clones resulted in germline transmission of the targeted allele, which was confirmed by Southern analysis.