The specific aims of the research described in this application are to identify, to isolate, and to biochemically and genetically characterize cell surface receptors (the """"""""phagocytosis receptors"""""""") which participate in the phagocytosis of photoreceptor outer segments (OS) by retinal pigment epithelial (RPE) cells. The long-term objectives of this research are to elucidate the biochemical and molecular events which control the phagocytosis of OS by RPE cells. Since RPE cells perform a number of functions which are crucial to the function and survival of photoreceptor cells, a mutation in any one of the functions may cause death of the underlying photoreceptor cells. Such a situation exists in the RCS rat, in which a recessive mutation greatly reduces the ability of RPE cells to phagocytize the shed tips of OS. Although the mutation is localized to the RPE cells, it is the underlying photoreceptors which degenerate. With RPE transplantation being studied as a possible treatment of diseases such as macular degeneration, a clear understanding of the interaction of photoreceptor cells with RPE cells will be essential for the successful culture, transplantation and survival of both cell types.
The specific aims will be achieved as follows. The investigator has identified a 55 kDa RPE plasma membrane glycoprotein (gp55) which, by a number of criteria, is a candidate for the phagocytosis receptor, and have isolated and sequenced a clone which encodes this protein from a rat RPE cDNA library. It shows no significant homology to any known protein. Polyclonal antibodies will be generated to gp55 which will be used to localize the protein in RPE cells by fluorescence microscopy and to inhibit the phagocytosis of OS by cultured RPE cells. The cDNA encoding this protein will be isolated from an RCS rat RPE cDNA library and sequenced in order to identify any mutations which could account for the phagocytosis defect. The complete gene sequence will also be obtained from a human genomic library. The normal and mutant cDNAs will be introduced into non-phagocytic eukaryotic cell lines, and the ability of these cells to phagocytize OS will be studied. Two other RPE glycoproteins (gp34 and gp36) are also candidates to participate in OS phagocytosis. These will be studied as described for gp55. The participation in OS phagocytosis of these three, and two other glycoproteins (mannose receptor and CD36) which have been proposed as candidates for the putative phagocytosis receptor, will be studied using antisense oligonucleotide technology and inhibition by specific antibodies. The ligands on rat OS, which are recognized by these putative receptors, will be isolated by affinity chromatography.
| Hall, Michael O; Agnew, Brian J; Abrams, Toshka A et al. (2003) The phagocytosis of os is mediated by the PI3-kinase linked tyrosine kinase receptor, mer, and is stimulated by GAS6. Adv Exp Med Biol 533:331-6 |
| Hall, Michael O; Abrams, Toshka A; Burgess, Barry L (2003) Integrin alphavbeta5 is not required for the phagocytosis of photoreceptor outer segments by cultured retinal pigment epithelial cells. Exp Eye Res 77:281-6 |
| Hall, Michael O; Obin, Martin S; Prieto, Anne L et al. (2002) Gas6 binding to photoreceptor outer segments requires gamma-carboxyglutamic acid (Gla) and Ca(2+) and is required for OS phagocytosis by RPE cells in vitro. Exp Eye Res 75:391-400 |
