RESUMO
BACKGROUND INFORMATION: αvß5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvß5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately. It is therefore unknown if αvß5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing αvß5 receptor levels affect POS binding and internalisation by wild-type (wt), αvß5- or MerTK-deficient RPE. RESULTS: ß5 integrin-green fluorescent protein (ß5-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 αvß5 receptors. In ß5(-/-) RPE cells, de novo formation of αvß5-GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvß5 surface receptors by over-expressing ß5-GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvß5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvß5 receptors of wt RPE cells. CONCLUSIONS: Expressing ß5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from ß5(-/-) 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 αvß5 and αvß5-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 αvß5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvß5 integrin receptors in RPE cells.