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Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches
SC Mueller, T Hasegawa, SS Yamada, KM Yamada and WT Chen
Department of Anatomy and Cell Biology, Georgetown University Medical School, Washington, DC 20007.
The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co- localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti- fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.
Volume 36,
Issue 3,
pp. 297-306,
03/01/1988
Copyright © 1988 by The Histochemical Society
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