Conclusions
Expressing β5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from β5(-/-) mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvβ5 and αvβ5-GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvβ5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvβ5 integrin receptors in RPE cells.
Results
β5 integrin-green fluorescent protein (β5-GFP) fusion proteins formed heterodimeric receptors with endogenous αv integrin subunits at the apical surface of mouse or rat RPE cells that co-immunoprecipitated focal adhesion kinase and redistributed with bound POS such as endogenous αvβ5 receptors. In β5(-/-) RPE cells, de novo formation of αvβ5-GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvβ5 surface receptors by over-expressing β5-GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvβ5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvβ5 receptors of wt RPE cells. Conclusions: Expressing β5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from β5(-/-) mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvβ5 and αvβ5-GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvβ5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvβ5 integrin receptors in RPE cells.