Abstract
Abstract: :
Purpose: PRL–1 (phosphatase of regenerative liver–1), originally identified as an immediate early gene in the regenerating liver, was detected in a search for fovea–associated genes. We are interested in determining the function of this small, isoprenylated protein tyrosine phosphatase in the human retina. To this end we have developed biotools (antibodies, recombinant proteins, and transfected cell lines) to localize PRL–l within the retina. Methods: PRL–1 mRNA expression was analyzed in 4 mm diameter punches of macula and mid peripheral human retina by quantitative RT–PCR. Recombinant wildtype and ‘substrate–trap’ mutant (C104S) PRL–1 proteins were expressed with and without green fluorescent protein tags and purified, and affinity–purified polyclonal antibodies were prepared. In vitro phosphatase activities of the recombinant PRL–1s were assayed using p–nitrophenyl phosphate (pNPP). Immunohistochemical localization was determined on cryosections of human, monkey and pig retina labeled with anti–PRL–1 or anti–red/green or blue opsin antibodies. Western blots of human macula or mid–peripheral retina and RPE tissue extracts partitioned into membrane and cytoplasm fractions were labeled with antibodies against PRL–1. Endogenous PRL–1 was isolated from human retinal lysates separated by FPLC over a Mono Q column, run on 2D–gels and identified by MALDI–TOF and ES–MS/MS. Results: PRL–1 mRNAs were 1.6–fold more abundant in the adult human macula compared to the peripheral retina. Anti–PRL–1 antibodies labeled the outer segments of red/green, but not blue, cone photoreceptors. The affinity–purified antibody, which reacted with recombinant PRL–1, detected a band of the predicted molecular weight (20 kD) in both the macula and peripheral sample, and was present in higher concentrations in the membrane fraction versus the cytoplasmic fraction. Assays of phosphatase activity demonstrated that the recombinant PRL–1 was active versus C104S mutant PRL–1,which was inactive. 2D plus ES–MS/MS analysis showed endogenous PRL–1 was 20 kD with a pI of 9. Conclusions:Our studies have demonstrated novel expression of PRL–1 in the neural retina where PRL–1 appears to be expressed in a subset of cones and is membrane–associated. Based on the cellular and subcellular localization of PRL–1 in the human retina, it may function in cone outer segment signal transduction, in a manner unrelated to its nuclear role in other tissues.
Keywords: photoreceptors • protein purification and characterization • signal transduction