Iacovelli L, Capobianco L, D'Ancona G M, Picascia A, De Blasi A
INM Neuromed, IRCCS, Località Camerelle, 86077 Pozzilli (IS), Italy.
J Endocrinol. 2002 Jul;174(1):103-10. doi: 10.1677/joe.0.1740103.
Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that activates a variety of biological activities including cell proliferation. Three mammalian LPA receptor (LPAr) subtypes have been identified by molecular cloning, named lp(A1), lp(A2) and lp(A3), that are coupled to heterotrimeric G-proteins for signal transduction. The LPAr are endogenously expressed in the rat thyroid cell line FRTL-5 and we used the FRTL-5 cells permanently transfected to obtain moderate overexpression of G-protein-coupled receptor kinase-2 (GRK2) or beta-arrestin1 to study whether GRK2 and beta-arrestin1 desensitise LPAr-mediated signalling and regulate LPA-stimulated functional effects. Using RT-PCR we documented that lp(A1), lp(A2) and lp(A3) receptors are all expressed in FRTL-5 cells. We then analysed the signal transduction of the LPAr in FRTL-5 cells. Exposure to LPA did not stimulate inositol phosphate formation nor cAMP accumulation but reduced forskolin-stimulated cAMP. LPA was also able to stimulate MAP kinase activation and this effect was abolished by pertussis toxin pretreatment. These results suggest that LPAr are mainly coupled to a pertussis toxin-sensitive G-protein in FRTL-5 cells. In order to investigate whether GRKs and arrestins are involved in the regulation of LPAr-mediated signalling, we used the FRTL-5 cell line permanently transfected to overexpress GRK2 (named L5GRK2 cells) or beta-arrestin1 (L5betaarr1 cells). The ability of LPA to inhibit forskolin-stimulated cAMP accumulation was blunted in L5GRK2 and more markedly in L5betaarr1. The MAP kinase activation was also blunted in L5GRK2 and in L5betaarr1B cells. Exposure to 20 microM LPA increased the phosphorylation of extracellular signal-regulated kinases ERK1/2 by approximately 3-fold in L5pBJI cells (FRTL-5 cells transfected with the empty vector pBJI) while it induced a modest increase in L5betaarr1 and was ineffective in L5GRK2. We measured [3H]thymidine uptake in L5betaarr1B and in L5 GRK2 cells to test whether GRK2 and beta-arrestin1 could have a role in the regulation of LPAr-mediated cell proliferation. The mitogenic response induced by 35 microM LPA was substantially blunted in L5betaarr1 (-69+/-6%) and in L5GRK2 (-69.8+/-4.5%) cells as compared with L5pBJI. Our findings document that the receptor-mediated responses elicited by LPA are regulated by GRK2 and beta-arrestin1 in FRTL-5 cells and indicate that this mechanism is potentially important for the control of the LPA-stimulated proliferative response.
溶血磷脂酸（LPA）是一种天然存在的磷脂，可激活多种生物活性，包括细胞增殖。通过分子克隆已鉴定出三种哺乳动物LPA受体（LPAr）亚型，分别命名为lp(A1)、lp(A2)和lp(A3)，它们与异源三聚体G蛋白偶联以进行信号转导。LPAr在大鼠甲状腺细胞系FRTL-5中内源性表达，我们使用永久转染的FRTL-5细胞来实现G蛋白偶联受体激酶-2（GRK2）或β-抑制蛋白1的适度过表达，以研究GRK2和β-抑制蛋白1是否使LPAr介导的信号脱敏并调节LPA刺激的功能效应。通过逆转录聚合酶链反应（RT-PCR），我们记录到lp(A1)、lp(A2)和lp(A3)受体均在FRTL-5细胞中表达。然后我们分析了FRTL-5细胞中LPAr的信号转导。暴露于LPA不会刺激肌醇磷酸的形成或环磷酸腺苷（cAMP）的积累，但会降低福斯可林刺激的cAMP。LPA还能够刺激丝裂原活化蛋白激酶（MAP激酶）的激活，并且这种效应被百日咳毒素预处理所消除。这些结果表明，在FRTL-5细胞中，LPAr主要与对百日咳毒素敏感的G蛋白偶联。为了研究GRK和抑制蛋白是否参与LPAr介导的信号调节，我们使用永久转染以过表达GRK2的FRTL-5细胞系（命名为L5GRK2细胞）或β-抑制蛋白1（L5βarr1细胞）。LPA抑制福斯可林刺激的cAMP积累的能力在L5GRK2细胞中减弱，在L5βarr1细胞中更明显。MAP激酶的激活在L5GRK2细胞和L5βarr1B细胞中也减弱。暴露于20微摩尔/升LPA可使细胞外信号调节激酶ERK1/2的磷酸化在L5pBJI细胞（用空载体pBJI转染的FRTL-5细胞）中增加约3倍，而在L5βarr1细胞中诱导适度增加，在L5GRK2细胞中无效。我们测量了L5βarr1B细胞和L5 GRK2细胞中[3H]胸腺嘧啶核苷的摄取，以测试GRK2和β-抑制蛋白1是否可能在LPAr介导的细胞增殖调节中发挥作用。与L5pBJI细胞相比，35微摩尔/升LPA诱导的促有丝分裂反应在L5βarr1细胞（-69±6%）和L5GRK2细胞（-69.8±4.5%）中显著减弱。我们的研究结果表明，LPA引发的受体介导反应在FRTL-5细胞中受GRK2和β-抑制蛋白1调节，并表明该机制对于控制LPA刺激的增殖反应可能很重要。
