Characterization of the interaction between protein 4.1R and ZO-2 - A possible link between the tight junction and the actin cytoskeleton

Multiple isoforms of the red cell protein 4.1R are expressed in nonerythroi d cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, w e found that two 4.1R isoforms of 135 and 150 kDa specifically interact wit h the protein ZO-2 (zonula occludens-2). 4.1R is colocalized with ZO-2 and occludin at Madin-Darby canine kidney (MDCK) cell tight junctions. Both iso forms of 4.1R coprecipitated with proteins that organize tight junctions su ch as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the pre sence of actin and cu-spectrin in these immunoprecipitates. Association of 4.1R isoforms with these tight junction and cytoskeletal proteins was found to be specific for the tight junction and was not seen in nonconfluent MDC K cells. The amino acid residues that sustain the interaction between 4.1R and ZO-2 reside within the amino acids encoded by exons 19-21 of 4.1R and r esidues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectr in/actin- and ZO-2-binding domains was recruited to tight junctions in conf luent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by est ablishing a link between the tight junction and the actin cytoskeleton.