RESUMO
Receptor protein tyrosine phosphatases (RPTPs) form a group of over 20 enzymes in vertebrates, each with unique ectodomains subject to potential extracellular interactions with ligands. It has recently become clear that a remarkably diverse range of ligands exist, including homophilic binders, adhesion molecules, neurotrophin receptors, and proteoglycans. Individual RPTPs can bind several ligands, and vice versa, suggesting that complex cell signaling networks exist. The identification of RPTP ligands and where they are located in tissues remains a challenge for a large number of these enzymes. Here we describe some powerful methods that have proved successful for several research groups, leading to our improved understanding of RPTP-ligand interactions and functional regulation.
Assuntos
Cromatografia de Afinidade/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura/métodos , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Isoenzimas/análise , Isoenzimas/metabolismo , Ligantes , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Ratos , Proteínas Tirosina Fosfatases Semelhantes a Receptores/análiseRESUMO
The activity and/or localization of receptor tyrosine kinases and phosphatases are controlled by binding to cell-surface or secreted ligands. Identification of ligands for receptor tyrosine phosphatases (RPTPs) is essential for understanding their in vivo functions during development and disease. Here we describe a novel in vivo method to identify ligands and binding partners for RPTPs by staining live-dissected Drosophila embryos. Live dissected embryos are incubated with RPTP fusion proteins to detect ligand binding in embryos. This method can be streamlined to perform large-scale screens for ligands as well as to search for embryonic phenotypes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Imuno-Histoquímica/métodos , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Animais , Membrana Celular , Dissecação/métodos , Drosophila/metabolismo , Proteínas de Drosophila/análise , Feminino , Ligantes , Masculino , Ligação Proteica , Proteínas Tirosina Fosfatases Semelhantes a Receptores/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.