Isoform Expression of Membrane Cofactor Protein (MCP; CD46) on Human Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: Membrane cofactor protein (MCP; CD46) is a complement regulatory protein widely expressed in nucleated cells as four isoforms (C1, C2, BC1 and BC2) that arise via alternative splicing. Two distinct cytoplasmic tails (CYT-1 and CYT-2) play pivotal roles in intracellular precursor processing and basolateral localization on polarized epithelium. We have previously shown that CD46 localizes on the basolateral surface of human retinal pigment epithelial (RPE) cells and associates with ß1 integrin. Antibody function-blocking experiments suggest that both CD46 and integrin play a role in RPE adhesion to Bruch’s membrane. This study is to investigate if the four isoforms are expressed in human RPE and to determine whether CD46 can be phosphorylated. Methods: Freshly isolated human RPE from donor eyes and ARPE-19 cells from human immortalized RPE were used for RT-PCR to identify CD46 isoforms. Both cDNA synthesis and PCR were performed from RNA using gene-specific primers and the Superscript One-Step RT-PCR system. Mature ARPE-19 cultures were treated with or without freshly prepared 10mM pervanadate, a phosphatase inhibitor for 10 minutes and lysed with 1% BRIJ lysis buffer, and immunoprecipitated with anti-CD46, anti-phosphotyrosine or control mAb. The precipitates were solubilized and eletrophoresed on SDS-PAGE and then transferred and blotted with a rabbit polyclonal anti-CD46 or with anti-phosphotyrosine. Results: All four isoforms were expressed by RPE cells from donor eyes and ARPE-19 cells. The BC2 isoform was the predominant isoform in human RPE and in mature ARPE-19 cultures maintained for four months. The BC1 isoform was predominant in immature ARPE-19 cultures maintained for only two weeks. Treatment of ARPE-19 cultures with pervanadate induced tyrosine phosphorylation and the phosphorylated bands were in the same molecular weight position as CD46. Protein tyrosine phosphorylation was not detected in RPE cells (without pervanadate) or in cells immunoprecipitated with a control mAb. Conclusions: Recent studies have shown that the CD46 cytoplasmic tail of the BC2 isoform can be phosphorylated on tyrosine. Our findings that the BC2 isoform is predominant in human RPE and mature ARPE-19 cells and that it can be tyrosine phosphorylated is suggestive of a possible signaling pathway for CD46 in RPE function.

Keywords: retinal pigment epithelium • protein structure/function • phosphorylation