Abstract
Purpose :
We have previously shown that βA3/A1-crystallin modulates lysosomal-mediated clearance in RPE cells. This study was undertaken to provide evidence that loss of this protein in RPE cells may also trigger abnormal endocytosis and affect intracellular signaling pathways in RPE cells.
Methods :
Cryba1 cKO mice lacking βA3/A1-crystallin specifically in the RPE, and age-matched floxed control mice were used in this study. In addition, we generated βA1 knockdown and βA3 knockout mice to determine if the Cryba1 gene products (βA1 and βA3-crystallin) have distinct functions in RPE cells. Pull down experiments were carried out to investigate the interaction between PTPN1/EGFR and βA3/A1-crystallin. QPCR and western blotting techniques were used to detect the expression of genes and proteins of interest, respectively, based on datasets obtained from RNAseq and phosphoproteomics analysis.
Results :
Co-IP experiments confirmed the interaction between βA3/A1-crystallin and EGFR in RPE cells. Reduced tyrosine phosphatase activity was detected in Cryba1 cKO RPE cells; Phosphoproteomics data suggested that the phosphorylation levels of proteins related to vesicle trafficking, such as FNBP1, BIN1, and DYN3 (key regulators in clathrin-coated vesicle budding and fission) were altered in cKO RPE cells compared to that of control mice. Also, we observed a decrease in Ezrin/Radixin and Rab5 (an early endosomal marker) expression in cKO RPE cells. RNAseq data showed up-regulation of the MAPK signaling pathway in the RPE of 3-month old Cryba1 cKO mice. Loss of either of the gene products of Cryba1 showed compensatory mechanisms between the proteins in the RPE.
Conclusions :
It can be concluded that βA3/A1-crystallin may play a multifaceted role in RPE cells, including involvement in EGFR/clathrin-dependent endocytosis and dephosphorylation, by regulating PTPN1 activity. Loss of this protein in RPE cells affects the normal endocytosis processes, thereby altering downstream signaling pathways, including the MAPK signaling pathway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.