Jiang H, Kuang Y, Wu Y, Smrcka A, Simon M I, Wu D
Department of Pharmacology, University of Rochester, Rochester, New York 14534, USA.
J Biol Chem. 1996 Jun 7;271(23):13430-4. doi: 10.1074/jbc.271.23.13430.
Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.
利用COS-7细胞中的共转染检测系统，对介导C5a和甲酰甲硫氨酸-亮氨酸-苯丙氨酸（fMet-Leu-Phe，fMLP）诱导的百日咳毒素（PTx）敏感的磷脂酶C（PLC）激活的信号转导通路进行了研究。由于在体外重组实验中已表明PLCβ2和PLCβ3均可被G蛋白的βγ亚基激活，因此测试了C5a和fMLP受体通过COS-7细胞内源性Gi蛋白的Gβγ亚基激活PLCβ2和PLCβ3的能力。在转染的COS-7细胞中，这两种受体均不能激活内源性PLCβ3或重组PLCβ3。然而，两种受体均能以PTx敏感的方式明显激活PLCβ2，这表明这些受体可能与内源性PTx敏感的G蛋白相互作用，并可能通过Gβγ亚基激活PLCβ2。共转染实验结果进一步证实了这些发现，即PLCβ3在共转染实验中仅能被Gβ1γ1或Gβ1γ5轻微激活，而在相同条件下Gβγ亚基能强烈激活PLCβ2。在共转染实验中，PLCβ3可被Gαq、Gα11和Gα16激活。此外，将C末端半胱氨酸残基替换为丝氨酸残基的Gγ2和Gγ3突变体能够抑制野生型Gβγ介导的PLCβ2激活，也能够抑制C5a或fMLP介导的PLCβ2激活。然而，这些Gγ突变体对由Gq类G蛋白介导的M1型毒蕈碱受体介导的PLC激活几乎没有影响。这些结果均证实Gβγ亚基参与了这两种趋化因子受体对PLCβ2的激活，并表明在COS-7细胞中，Gβγ对PLCβ3的激活可能不是这些受体的主要途径。
