The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site
The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site
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DOI：
10.1021/bi971765v
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发表时间：
1998-01-06
期刊：
影响因子：
2.9
通讯作者：
Villalobo, A
中科院分区：
文献类型：
--
作者：
Martín-Nieto, J;Villalobo, A
通过钙离子依赖性钙调素亲和色谱纯化富集配体不敏感形式的人表皮生长因子受体（EGFR）。碱性两亲片段Arg（645）-Arg-Arg-His-Ile-val-Arg-Lys-Arg-Thr（654）-Leu-Arg-Arg-Leu-Leu-Gln（660），位于该受体的胞质质膜结构域内，被纯化为与谷胱甘肽S-转移酶的融合蛋白，并显示以Ca 2+依赖性方式结合钙调蛋白。测定了该结合过程的表观解离常数为0.3 μ M钙调素（K-d（CaM β ′））和表观亲和常数为0.5 μ M游离Ca ~（2+）（K-a（CaM β ′））。钙调素在质膜位点的结合阻止了蛋白激酶C对Thr-654残基的磷酸化，并测定了0.5-1 μ M钙调素（K-1（CaM β ′））的表观抑制常数。相反，蛋白激酶C磷酸化该位点阻止了其随后与钙调蛋白的相互作用。因此，我们提出，由钙调蛋白和蛋白激酶C介导的信号通路之间的串扰发生在EGFR的质膜结构域。该钙调素结合序列在脊椎动物EGFR家族的蛋白酪氨酸激酶中高度保守。
A ligand-insensitive form of the human epidermal growth factor receptor (EGFR) was enriched by Ca2+-dependent calmodulin-affinity chromatography purification. The basic amphiphilic segment Arg(645)-Arg-Arg-His-Ile-val-Arg-Lys-Arg-Thr(654)-Leu-Arg-Arg-Leu-Leu-Gln(660), located within the cytoplasmic juxtamembrane domain of this receptor, was purified as a fusion protein with glutathione S-transferase and shown to bind calmodulin in a Ca2+-dependent manner. An apparent dissociation constant of 0.3 mu M calmodulin (K-d(CaM)') and an apparent affinity constant of 0.5 mu M free Ca2+ (K-a(Ca)') were measured for this binding process, Binding of calmodulin at the juxtamembrane site prevented the phosphorylation of residue Thr-654 by protein kinase C, and an apparent inhibition constant of 0.5-1 mu M calmodulin (K-i(CaM)') was determined. Conversely, phosphorylation of this site by protein kinase C prevented its subsequent interaction with calmodulin. We therefore propose that cross talk between signaling pathways mediated by calmodulin and protein kinase C occurs at the juxtamembrane domain of the EGFR. This calmodulin-binding sequence highly conserved among protein tyrosine kinases of the vertebrate EGFR family.