Mastrangelo A M, Homan S M, Humphries M J, LaFlamme S E
Department of Physiology and Cell Biology Albany Medical College, Albany, NY 12208, USA.
J Cell Sci. 1999 Jan;112 ( Pt 2):217-29. doi: 10.1242/jcs.112.2.217.
The role of beta cytoplasmic domains in regulating beta1 integrin conformation and function in cell attachment is not fully understood. In this study, we tested the ability of transiently expressed beta cytoplasmic domains connected to an extracellular reporter domain to regulate 'in trans' the conformation of endogenous beta1 integrins, and compared these effects on cell attachment. We found that chimeric receptors containing either the beta1, beta3 or beta5 cytoplasmic domains inhibited the expression of the conformationally dependent 9EG7 and 12G10 epitopes on endogenous beta1 integrins. In contrast, chimeric receptors containing the beta4 or alpha5 cytoplasmic domain, or a control receptor lacking a cytoplasmic domain, had no effect. This inhibition occurred in a dose-dependent manner that required high levels of expression of the chimeric receptor. These results suggest that beta1 integrin conformation can be regulated by conserved cytosolic interactions involving beta cytoplasmic domains. This is further supported by our findings that mutations within amino acid motifs conserved among these beta cytoplasmic domains, specifically the NXXY, NPXY and TST-like motifs, reduced the ability of these chimeric receptors to regulate beta1 integrin conformation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST-like motifs. The beta1 chimera also inhibited the binding of soluble fibronectin to endogenous beta1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9EG7 and 12G10 epitopes correlates with integrin function. However, Mn2+, which is an extracellular activator of integrin function, increased 9EG7 expression to basal levels in the presence of the beta1 chimera, but did not rescue cell attachment to the same extent. Thus, although the beta1 integrin conformation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the beta chimeras may be inhibiting both ligand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attachment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function. This finding, together with the requirement for high levels of the chimeric receptors and the fact that mutations in the same conserved motifs in heterodimeric beta1 integrins have been reported to regulate beta1 integrin conformation and function in cell attachment, suggest that beta cytoplasmic domains regulate these processes by interacting with cytosolic factors and that the regulatory effect of the chimeras may be due to their ability to titrate proteins from endogenous integrins.
β细胞质结构域在调节β1整合素构象及细胞黏附中的功能尚未完全明确。在本研究中，我们测试了与细胞外报告结构域相连的瞬时表达β细胞质结构域调节内源性β1整合素“反式”构象的能力，并比较了它们对细胞黏附的影响。我们发现，含有β1、β3或β5细胞质结构域的嵌合受体抑制了内源性β1整合素上构象依赖性9EG7和12G10表位的表达。相反，含有β4或α5细胞质结构域的嵌合受体，或缺乏细胞质结构域的对照受体则没有影响。这种抑制以剂量依赖性方式发生，需要嵌合受体的高水平表达。这些结果表明，β1整合素构象可通过涉及β细胞质结构域的保守胞质相互作用来调节。我们的研究结果进一步支持了这一点，即在这些β细胞质结构域中保守的氨基酸基序内的突变，特别是NXXY、NPXY和TST样基序，降低了这些嵌合受体调节β1整合素构象的能力。有趣的是，嵌合受体以类似的剂量依赖性方式抑制细胞黏附，并且需要完整的NXXY、NPXY和TST样基序。β1嵌合体还抑制了可溶性纤连蛋白与内源性β1整合素的结合。因此，嵌合体对构象依赖性整合素表位表达、细胞黏附和配体结合的同时抑制表明，9EG7和12G10表位的表达与整合素功能相关。然而，作为整合素功能细胞外激活剂的Mn2 +，在存在β1嵌合体的情况下将9EG7表达增加到基础水平，但没有在相同程度上挽救细胞黏附。因此，尽管单克隆抗体9EG7识别的β1整合素构象可能是细胞黏附所必需的，但并不充分，表示β嵌合体可能同时抑制细胞黏附所需的配体结合和配体结合后事件。此外，嵌合受体对细胞黏附的抑制作用不会因添加先前已证明可抑制整合素功能的细胞内信号的药物而逆转。这一发现，连同对高水平嵌合受体的需求以及据报道异二聚体β1整合素中相同保守基序的突变可调节细胞黏附中βl整合素构象和功能这一事实，表明β细胞质结构域通过与胞质因子相互作用来调节这些过程，并且嵌合体的调节作用可能归因于它们从内源性整合素中滴定蛋白质的能力。
