Tahiliani P D, Singh L, Auer K L, LaFlamme S E
Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208, USA.
J Biol Chem. 1997 Mar 21;272(12):7892-8. doi: 10.1074/jbc.272.12.7892.
Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin beta1, beta3, and beta5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, beta3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin beta3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the beta3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the beta3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the beta3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the beta3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the beta3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the beta3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.
整合素介导的细胞与细胞外基质蛋白的黏附会触发多种细胞内信号通路，包括一系列酪氨酸磷酸化反应。在许多细胞类型中，细胞质粘着斑酪氨酸激酶FAK似乎是响应黏附而首先发生酪氨酸磷酸化的蛋白质；然而，调节整合素触发的FAK磷酸化的分子机制尚不清楚。先前的研究表明，整合素β1、β3和β5亚基的细胞质结构域在与细胞外报告基因相连的单亚基嵌合受体中表达时，都包含足以触发FAK磷酸化的信息。在本研究中，构建了β3细胞质结构域缺失和替代突变体，以鉴定整合素β3细胞质结构域内调节其触发FAK磷酸化能力的氨基酸。对瞬时表达含有这些突变细胞质结构域的嵌合受体的细胞进行磁性分选，并检测FAK的酪氨酸磷酸化。对这些突变体的分析表明，β3细胞质结构域的膜近端和C末端片段中的结构信息对于触发FAK磷酸化很重要。在β3细胞质结构域的C末端片段中，发现高度保守的NPXY基序是β3细胞质结构域触发FAK磷酸化所必需的。然而，发现β3细胞质结构域N末端片段内的假定FAK结合结构域对于该信号事件既不是必需的也不是充分的。我们还证明，已知导致Glanzmann血小板无力症变体的丝氨酸752到脯氨酸突变会抑制β3细胞质结构域信号传导FAK磷酸化的能力，这表明β3细胞质结构域中的单个突变可以抑制“由内向外”和“由外向内”的整合素信号传导。
