Identification and localization of a beta-1 receptor from the integrin family in mammalian retinal pigment epithelial cells.
The distribution of an adhesion receptor from the integrin family was mapped in cultured mammalian retinal pigment epithelial cells (RPE), and in primate RPE cells in vivo, using antibodies to the human fibronectin receptor (FnR) in conjunction with indirect immunofluorescence and immunoelectron microscopy. Protein homogenates from human RPE or MG-63 cells were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. On Western blots of proteins from both cell types, anti-FnR and a monoclonal antibody to the beta 1 subunit of human FnR each recognized a single band with a molecular mass of approximately 115 kDa. After 1-6 weeks in culture human, monkey and feline RPE cells gradually acquired a morphology and cytoskeletal arrangement typical of a mature cuboidal epithelium. The distribution of anti-FnR labeling changed dramatically in accordance with the cells' phenotype. In preconfluent cells, labeling consisted of streaks and flecks of fluorescence at the termini of stress fibers and at putative sites of cell substratum attachment. As the cells became confluent and acquired an epithelioid morphology, the bulk of anti-FnR labeling shifted to the peripheral cytoplasm and appeared as a cross-hatched meshwork. In fully differentiated RPE cells anti-FnR labeling consisted of a dense punctate pattern on or close to the apical cell surface that coincided with the distribution of f-actin as shown by phalloidin staining, and to the distribution of apical microvilli as identified by scanning electron microscopy. In addition, a compacted rim of fluorescence appeared at the cells' lateral margins that was also virtually identical to the phalloidin staining pattern. No basal surface labeling was apparent at this stage. At the ultrastructural level, FnR was localized to the apical surface of nonpermeabilized RPE cells and, in particular, to the plasma membrane of apical microvilli in vitro and in vivo using an indirect, pre-embedding method. The results strongly suggest that: (1) a membrane receptor/s containing the integrin beta 1 subunit is normally present on the plasma membrane of apical microvilli and on the lateral cell surfaces of cultured mammalian RPE cells; and (2) this receptor also is present on the plasmalemma of RPE apical microvilli in vivo.
Anderson
D H
DH
Neuroscience Research Institute, University of California, Santa Barbara 93106.
Guérin
C J
CJ
Matsumoto
B
B
Pfeffer
B A
BA
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002415:Cats; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D000882:Haplorhini; D006801:Humans; D007124:Immunoenzyme Techniques; D016023:Integrins / Q000032:analysis*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000032:analysis* / Q000648:ultrastructure; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000032:analysis*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D016023:Integrins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic