Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of αvβ5 integrin

Background information av beta 5 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 av beta 5 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 av beta 5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing av beta 5 receptor levels affect POS binding and internalisation by wild-type (wt), av beta 5- or MerTK-deficient RPE. Results beta 5 integringreen fluorescent protein (beta 5GFP) fusion proteins formed heterodimeric receptors with endogenous av 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 av beta 5 receptors. In beta 5-/- RPE cells, de novo formation of av beta 5GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of av beta 5 surface receptors by over-expressing beta 5GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in av beta 5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous av beta 5 receptors of wt RPE cells. Conclusions Expressing beta 5GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from beta 5-/- 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 av beta 5 and av beta 5GFP 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 av beta 5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface av beta 5 integrin receptors in RPE cells.