Abstract
Abstract: :
Purpose: Focal adhesion kinase (FAK)–dependent signaling promotes phagocytic engulfment of surface–tethered photoreceptor outer segment fragments (OS) by RPE cells. This study explores the signal transduction pathway downstream of FAK that links OS interaction with αvß5 integrin receptors to Mer tyrosine kinase (MerTK) activation. Methods: Primary RPE cultures were prepared from 12 to 15 day old Long Evans and RCS (MerTK mutant) rats. Permanent rat RPE–J cells were differentiated to a polarized epithelial monolayer. RPE cells were incubated with pharmacological inhibitors of tyrosine kinase cell signaling pathways or were induced to transiently express dominant negative mutants of signaling proteins. RPE cells were fed with isolated OS for different periods of time before analysis of signaling proteins using protein domain– and activity–specific antibodies. Laser scanning confocal microscopy of immunolabeled samples served to follow localization and recruitment of relevant proteins to sites of phagocytic activity. Cell fractionation, immunoprecipitation and immunoblotting were used to identify signaling protein complex formation and anchorage. Results: Long Evans primary rat RPE cells and stable rat RPE–J cells shared expression of the phagocytic receptors MerTK, CD36, and αvß5 integrin. These different in vitro models of RPE responded similarly to OS phagocytic challenge with αvß5 integrin redistribution to nascent phagocytic cups at the apical surface. These RPE cells also shared expression of Src kinase family members and of proteins containing SH2 domains that recognize changes in protein tyrosine phosphorylation. The adaptor protein Grb2, c–Src and Lyn kinases resided in a signaling complex with FAK and showed increased tyrosine phosphorylation in response to OS phagocytic challenge. However, the downstream targets of FAK signaling differed from each other in kinetics of phosphorylation, time of residence in a complex with FAK, and in sensitivity to FAK and Src inhibition. Conclusions: These experiments identify further elements of an important phagocytic signaling pathway via αvß5 integrin and FAK that is initiated by RPE cells in response to OS challenge. The distinct activation characteristics of individual members of the Src kinase family suggests that they function at different steps of this signaling cascade.
Keywords: signal transduction • retinal pigment epithelium • phosphorylation