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S.C. Finnemann, Y. Chang; Regulation of RPE Phagocytosis by Integrin Receptor–Tetraspanin Surface Membrane Domains . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5879.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cells depend on αvß5 integrin receptors and their downstream signaling pathways to maximize their phagocytic activity following circadian photoreceptor outer segment (POS) shedding in the retina. We hypothesize that the activity of αvß5 integrin itself may be regulated in the RPE to synchronize phagocytosis. Integrin receptors may associate with proteins of the tetraspanin family in specialized membrane microdomains to control integrin signaling. Here, we test whether tetraspanins functionally interact with αvß5 integrin in RPE cells.
RT–PCRs and immunoblotting established tetraspanin expression in RPE cells in vitro and in vivo. We used confocal microscopy following live cell labeling with receptor antibodies and choleratoxin B to co–localize αvß5 integrin and tetraspanins to plasma membrane sub–domains at the apical surface of the RPE. We used optiprep gradient fractionation of RPE cell lysates and co–immunoprecipitation assays to further test subcellular co–fractionation of tetraspanins and αvß5 integrin. We used isolated POS fragments in quantitative uptake assays to determine effects of surface cholesterol depletion, tetraspanin antibody blocking and tetraspanin overexpression on the phagocytic function of RPE cells in culture.
We found that tetraspanins CD9, CD63 and CD81 localize to the apical, phagocytic surface of RPE cells in culture. CD81 partially co–localized with αvß5 integrin. Furthermore, we identified CD81 in a complex with αvß5 integrin in RPE cells in culture and in intact retina. CD81 co–fractionated with αvß5 in low–density membrane domains of RPE cell lysates. Cholesterol depletion, which altered CD81 and αvß5 surface distribution, as well as specific inhibition of CD81 reduced POS phagocytosis by human and rat RPE cell cultures. In contrast, CD81 overexpression increased POS phagocytosis.
Our results demonstrate that the apical plasma membrane tetraspanin CD81 regulates the phagocytic activity of the RPE. Functional interaction with tetraspanins including CD81 and microdomain recruitment may contribute to the temporal control of αvß5 integrin receptor signaling in the RPE that is necessary for rhythmic phagocytosis of shed POS. Ongoing experiments study CD81 and integrin αvß5 complex formation and distribution during active RPE phagocytosis in vitro and in vivo.
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