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E.A. Pierce, A. Saveliev, Q. Liu; The Retinitis Pigmentosa 1 (RP1) Protein Interacts With C–Jun N–Terminal Kinase–Interacting Proteins (JIPs) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3727.
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Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP). The mechanism by which mutations in RP1 lead to photoreceptor death and the function of RP1 protein are only starting to become understood. We recently reported the RP1 protein is a photoreceptor–specific microtubule associated protein (MAP), and that the MAP functions of RP1 are mediated by N–terminal doublecortin domains. Disruption of the Rp1 gene in mice causes disorganization of the membrane discs in the outer segments of photoreceptor cells. To help define the mechanism by which RP1 helps control outer segment organization, we are using yeast–two hybrid analyses to identify proteins that interact with RP1.
Using a GAL4–based yeast two–hybrid system, we screened a mouse retinal cDNA library with 4 different portions of the mouse Rp1 cDNA (N–Rp1 241–661aa, M1–Rp1 555–1013aa, M2–Rp1 1079–1535aa, C–Rp1 1393–2088aa,) as baits. Positive yeast–two hybrid colonies were isolated and verified by specificity testing. The potential interaction between Rp1 and candidate proteins was further evaluated by co–expression and co–immunoprecipitation assays using cultured cells and retinal extracts. Co–localization of candidate interacting proteins with Rp1 was examinated by immunostaining assays.
A total of 32 specific clones were identified with baits N–Rp1 and M1–Rp1. Of these, 15 encode JNK–interacting proteins (Jips). Co–immunoprecipitation of Jip–1 and Rp1 demonstrate that recombinant N–Rp1 and Jip–1 interact in cultured cells. Rp1 and Jip–1 proteins also co–immunoprecipitate each other from mouse retinal extracts. Finally, Rp1 and Jip–1 co–localize in the axonemes of photoreceptor outer segments. Evaluation of additional Rp1 candidate interacting proteins is in progress.
We have identified JIP–1 as an RP1 interacting protein by yeast two hybrid analysis. JIP proteins are thought to act as molecular scaffolds that organize the c–Jun N–terminal kinase (JNK) and other signal transduction pathways in response to specific stimuli. JIP proteins also interact with the light chain of kinesin I. We therefore hypothesize that like other MAPs such as doublecortin and MAP2, RP1 function may be regulated by phosphorylation via JNK, which in turn is associated with RP1 via a JIP scaffold protein. Additional experiments are in progress to determine the physiologic consequences of this Jip–Rp1 interaction.
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