Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.
Bottom Line: The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs.The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly.Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.
Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.Show MeSH
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Mentions: We have exploited the fact that mouse GFP-talin1 can be used to rescue the talin1 knockdown phenotype in subconfluent HUVEC to carry out a comprehensive structure function study on talin1 in a cellular context (Fig. 7A). We started by testing the effects of three mutations in the talin F3 FERM domain that have been shown to compromise integrin binding or activation (Garcia-Alvarez et al., 2003; Wegener et al., 2007). Previous analysis of such mutations has largely been restricted to monitoring effects on integrin activation, and not cellular responses such as cell spreading or FA assembly. Surprisingly, expression of the GFP-talin1 R358A mutant largely rescued the talin1 knockdown phenotype in HUVEC, and the same mutant has recently been shown to support Mn2+-induced αVβ3-integrin clustering in B16 melanoma cells (Saltel et al., 2009), and integrin clustering in muscle (Tanentzapf and Brown, 2006). The structure of F3 bound to the β3-integrin tail shows that R358 is part of a pocket that accommodates W739 of the β3-tail (Garcia-Alvarez et al., 2003). The guanadinium group of Arg358, which stacks parallel to the aromatic ring of W739 in the β3-tail, points into solution, and it is likely that its loss would not perturb the rest of the pocket or the structure of the F3 domain. Therefore, although the R358A mutation totally ablated binding of a β3-tail peptide to F2F3 in vitro, it is likely that in the context of full-length talin expressed in the cell, other interactions between the talin head, integrin tails and acidic membrane phospholipids (Goult et al., 2010; Anthis et al., 2009; Saltel et al., 2009) partially compensate for the effect of the R358A mutation on integrin binding.
Affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.