Luttrell L M, Hawes B E, Touhara K, van Biesen T, Koch W J, Lefkowitz R J
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1995 Jun 2;270(22):12984-9. doi: 10.1074/jbc.270.22.12984.
Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.
普列克底物蛋白同源（PH）结构域是蛋白质序列同源性的90 - 110个氨基酸区域，存在于多种参与信号转导和生长控制的蛋白质中。我们之前报道过，几种蛋白质的PH结构域，包括β - ARK1、PLCγ、IRS - 1、Ras - GRF和Ras - GAP，以谷胱甘肽S - 转移酶融合蛋白形式表达时，能在体外以不同亲和力可逆地结合纯化的牛脑Gβγ亚基。为了确定PH结构域肽在完整细胞中是否会作为Gβγ亚基介导的信号转导的拮抗剂，制备了编码这些PH结构域的质粒小基因构建体，其允许肽在细胞中瞬时表达。在瞬时共表达α2 - C10肾上腺素能受体（AR）和每种PH结构域肽的COS - 7细胞中，百日咳毒素敏感的、Gβγ亚基介导的肌醇磷酸（IP）产生被显著抑制。百日咳毒素不敏感的、通过共表达的M1毒蕈碱型乙酰胆碱受体（M1 AChR）介导的Gqα亚基介导的IP产生仅被PLCγ PH结构域肽减弱，这表明大多数PH结构域肽的抑制作用是Gβγ亚基特异性的。据报道，COS - 7细胞中Gi偶联受体对丝裂原活化蛋白（MAP）激酶途径的刺激需要p21ras的激活且不依赖于蛋白激酶C。由于参与激活的几种蛋白质含有PH结构域，因此确定了PH结构域肽表达对α2 - C10 AR介导的p21ras - GTP交换和MAP激酶激活以及直接的Gβγ亚基介导的MAP激酶激活的影响。在每个实验中，PH结构域肽的共表达导致显著抑制。增加Gβγ亚基表达克服了PH结构域肽介导的对MAP激酶激活的抑制。这些数据表明，PH结构域肽在完整细胞中作为Gβγ介导信号转导的特异性拮抗剂，并且PH结构域与Gβγ亚基或结构相关蛋白之间的相互作用可能在G蛋白偶联受体激活有丝分裂信号通路中起作用。
