Identification of Conserved Residues Responsible for the Regulation of F‐actin Binding and Dimerization of the I/LWEQ Module of Talin1

The I/LWEQ module is an ancient, conserved C‐terminal actin‐binding element found in animal talins, Hip1/12, and fungal Sla2. The module has a consensus length of 186 amino acids arranged in four conserved blocks. Dysregulation of the expression of I/LWEQ module proteins has several negative consequences. Talin1 mutants lacking the I/LWEQ module do not form a linkage between the extracellular matrix and actin cytoskeleton. Overexpression of Hip12 mutants without the I/LWEQ module displays a similar phenotype to Hip12‐null cells, where clathrin‐coated vesicles accumulated due to delayed or blocked internalization. Yeast Sla2 lacking the I/LWEQ module results in abnormal endocytosis. These results indicate that the I/LWEQ module forms a link between the actin cytoskeleton and other cellular compartments, such as the plasma membrane and endocytic vesicles. We have previously shown that the I/LWEQ module contains a dimerization motif and that the C‐terminal Block 4 is essential for both dimerization and F‐actin binding. We have used site‐directed mutagenesis of Block 4 of mouse Talin1 to determine whether dimerization and actin binding are coupled in the I/LWEQ module. The mutant I/LWEQ modules displayed a significant decrease in F‐actin binding affinity. We used gel filtration chromatography, cross‐linking, and analytical ultracentrifugation to correlate actin binding with dimerization. Our results show that the regulation of dimerization and actin binding of the I/LWEQ module are coupled. Future research will examine the mechanisms of F‐actin binding and the role of the dimerization motif in I/LWEQ module‐containing proteins. NIH COBRE P20RR20171. AHA 0365218B.