Colocalization of GP125/CD98 with Tropomyosin Isoforms at the Cell-Cell Adhesion Boundary

Abstract

Two monoclonal antibodies designated as 1F6 and 4B10 were obtained on screening for reactivities to CD98-associated molecules by sandwich-type enzyme-linked immunosorbent assaying using hybridoma culture supernatants as the solid phase, cell lysates as an antigen source, and a mixture of biotinylated antibodies to CD98HC as a detector. Flow cytometric analysis with microspheres in combination with 1F6, 4B10, and anti-CD98HC also indicated the association of antibody-defined antigen(s) with CD98. 1F6 and 4B10, stained fibrillate components in fixed and permeated cells but were not reactive with unfixed live cells, suggesting that epitopes reside in the cytoskeleton-associated structure in the intracellular region. Two-color immunostaining followed by confocal microscopy revealed the colocalization of the antigen with CD98 at the cell-cell adhesion boundary of HeLa cells. 1F6 detected proteins with relative molecular masses of 33,000 to 43,000 on immunoblotting analysis involving cell lysates of human and rat cell lines. Analysis with a purified tropomyosin specimen from rabbit skeletal muscle demonstrated that 1F6 and 4B10 recognize tropomyosin. Two-dimensional gel electrophoresis followed by immunoblotting analysis revealed that 1F6 recognizes various tropomyosin isoforms. These results indicated that CD98 physically associates directly or indirectly with tropomyosin, and that this association is closely related to the cell-cell interaction.