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 nonerythroid cells, including novel 135-kDa isoforms. Using a yeast two-hybrid system, immunocolocalization, immunoprecipitation, and in vitro binding studies, we found that two 4.1R isoforms of 135 and 150 kDa specifically interact with 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 isoforms of 4.1R coprecipitated with proteins that organize tight junctions such as ZO-2, ZO-1, and occludin. Western blot analysis also revealed the presence of actin and alpha-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 MDCK 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 residues 1054-1118 of ZO-2. Exogenously expressed 4.1R containing the spectrin/actin- and ZO-2-binding domains was recruited to tight junctions in confluent MDCK cells. Taken together, our results suggest that 4.1R might play an important role in organization and function of the tight junction by establishing a link between the tight junction and the actin cytoskeleton.
Mattagajasingh
S N
SN
Department of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Huang
S C
SC
Hartenstein
J S
JS
Benz
E J
EJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton; D004285:Dogs; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009479:Neuropeptides; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013049:Spectrin / Q000378:metabolism; D019108:Tight Junctions; D020798:Two-Hybrid System Techniques; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D000199:Actins; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D062793:Occludin; D010750:Phosphoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin; D049452:Green Fluorescent Proteins