G_(12)亚型G蛋白对M_3乙酰胆碱受体介导的磷脂酶D调控

Regulation of M_3 muscarinic acetylcholine receptor-mediated PLD stimulation by G_(12)-type G proteins

目的:阐明何种Gα亚型蛋白偶联M3乙酰胆碱受体(M3mAChR)并介导PLD的激活。方法:将带有Gαq、Gα12及Gα13基因的质粒转染入HEK293细胞,在细胞内过度表达,用免疫杂交检测相应的表达蛋白,并测定胞内M3mAChR介导的PLD及PLC活性。结果:Gα12、Gα13亚型野生型及功能增强型蛋白的过度表达,导致胞内M3mAChR介导的PLD活性明显升高(P<0.001);而过度表达Gα12、Gα13功能缺陷型蛋白,则使胞内M3mAChR介导的PLD活性明显降低(P<0.001);但Gαq野生型及功能增强型蛋白的过度表达仅影响胞内PLC活性,对M3mAChR介导的PLD激活没有影响。结论:M3mAChR介导的胞内PLD激活极有可能是Gα12、Gα13而不是Gαq亚型G蛋白。

Objective： To determine the type of G protein mediating M3 mAChR-PLD coupling in comparison to M3 mAChR-PLC coupling. Methods ：we transiently transfected HEK-293 cells with empty expression vectors and expression plasraids for wild-type, constitutively active or dominant-negative mutants of Gα12, Gα13, Gαq, respectively. After 48 h, PLD and PLC activities were determined by measuring accumulation of [^3H] PtdEtOH and [^3H]IPx in the absence and presence of 1 mM carbachol, a typical agonist of M3 mAChR. Expression of the individual Get subunits were examined by immunobloting with their specific antibodies. Results：In HEK293 cells overexpressing wild-type either Gα12 or Gα13proteins, the PLD response to carbachol was increased by 2-fold compared to control cells, leaving basal PLD activity unaffected, although overexpression of wildtype either Gα12 or Gα13 did not modify M3 mAChR signaling to PLC. Intriguingly, the Gαq proteins potentiated greatly the PLC response to M3 mAChR. Furthermore, the similar（more dramatic） effect on PLD and PLC coupling to M3 mAChR compared to their wild-type proteins, was induced by constitutatively active mutants, Q229L Gα12, Q226L Gα13 and R183C Gαq. In contrast, overexpression of dominant-negative mutants G228A Gα12 and G225A Gα13 inhibited the PLD stimulation by M3 mAChR to a similar extent, around 50%, but no effect on PLD basal activity. Conclusions：These data suggested that the M3mAChR expressed in HEK-293 cells signals to PLD via G12 but not Gq-type G proteins.