Identification of a structural element in phospholipase C β2 that interacts with G protein βγ subunits
Identification of a structural element in phospholipase C β2 that interacts with G protein βγ subunits
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DOI：
10.1074/jbc.273.12.7148
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发表时间：
1998-03-20
影响因子：
4.8
通讯作者：
Smrcka, AV
中科院分区：
文献类型：
--
作者：
Sankaran, B;Osterhout, J;Smrcka, AV
为了描绘参与 G 蛋白 β γ 亚基结合和激活的磷脂酶 C β 2 (PLC β 2) 的特定区域，我们合成了与 PLC β 2 片段相对应的肽，对应于 Asn-564-Lys-583 (N20K) 和 Glu-574-Lys-593 (E20K) 的两个重叠肽抑制了 β γ 亚基对 PLC β 2 的激活 (IC50)分别为 50 和 150 μM），而两种对照肽（N20K 和 E20K，但对照肽除外）不会通过百日咳毒素和磷酸肌醇 3-激酶的 β γ 依赖性激活来抑制 G α(i1) 的 β γ 依赖性 ADP 核糖基化。为了证明肽与 β γ 亚基的直接结合，将肽与纯化的 β(1) 进行化学交联伽马(2)。 N20K 和 E20K 与 β(1) 和 γ(2) 亚基交联，而对照肽则不然。与过量 PLC β 2 或 PLC β 3 一起孵育可抑制与 β 和 γ 的交联，而与 γ 的交联则被 r-myr-alpha(i1) 抑制，但不与 β 交联。这些数据共同证明了 N20K 和 E20K 对 G β γ 结合和抑制 β γ 亚基效应子激活的特异性。结果表明，两种活性肽 Glu-574-Lys-583 的重叠区域模拟了参与与 β γ 亚基结合的 PLC β 2 区域。将肽的该重叠区域中的酪氨酸改为谷氨酰胺抑制了肽与 β γ 亚基的结合，这些肽与 PLC delta 1 的三维结构的比对确定了 PLC beta 2 催化结构域表面上的一个假定的 α 螺旋区域，该区域可以与 β γ 亚基相互作用。
To delineate the specific regions of phospholipase C beta 2 (PLC beta 2) involved in binding and activation by G protein beta gamma subunits, we synthesized peptides corresponding to segments of PLC beta 2, Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta 2 by beta gamma subunits (IC50 50 and 150 mu M, respectively), whereas two control peptides did not, N20K and E20K, but not the control peptides, inhibited beta gamma-dependent ADP-ribosylation of G alpha(i1) by pertussis toxin and beta gamma-dependent activation of phosphoinositide 3-kinase, To demonstrate direct binding of the peptides to beta gamma subunits, the peptides were chemically cross-linked to purified beta(1) gamma(2). N20K and E20K cross-linked to both beta(1) and gamma(2) subunits, whereas the control peptides did not, Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta 2 or PLC beta 3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alpha(i1). These data together demonstrate specificity of N20K and E20K for G beta gamma binding and inhibition of effector activation by beta gamma subunits, The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta 2 that is involved in binding to beta gamma subunits, Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to beta gamma subunits, Alignment of these peptides with the three-dimensional structure from PLC delta 1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta 2 that could interact with beta gamma subunits.