AFADIN - A NOVEL ACTIN FILAMENT-BINDING PROTEIN WITH ONE PDZ DOMAIN LOCALIZED AT CADHERIN-BASED CELL-TO-CELL ADHERENS JUNCTION

A novel actin filament (F-actin)-binding protein with a molecular mass of similar to 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat br ain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016-1,100 aa residues, a domain known to interact with transmembrane proteins. p20 5 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculate d molecular mass of 188,971 kD. p190 was a splicing variant of p205 ha ving one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin -binding domain. Homology search analysis revealed that the aa sequenc e of p190 showed 90% identity over the entire sequence with the produc t of the AF-6 gene, which was found to be fused to the ALL-1 gene, kno wn to be involved in acute leukemia. p190 is likely to be a rat counte rpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Wester n blot analyses showed that p205 was ubiquitously expressed in ail the rat tissues examined, whereas p190 was specifically expressed in brai n. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of variou s tissues. We named p205 l-afadin (a large splicing variant of AF-6 pr otein localized at adherens junction) and p190 s-afadin (a small splic ing variant of l-afadin). These results suggest that l-afadin serves a s a linker of the actin cytoskeleton to the plasma membrane at cell-to -cell AJ.