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D.C. Pentia, R.H. Cote; Characterization of Rod Photoreceptor Glutamic Acid Rich Protein Interaction with Rod Photoreceptor PDE6 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3180.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the interaction and functional significance of binding of the Glutamic Acid Rich Protein-2 (GARP2) to mammalian rod PDE6. Methods:Rod outer segments (ROS) were purified on a sucrose gradient. Proteins released by hypotonic treatment of ROS membranes were fractionated by hydrophobic interaction chromatography (HIC). The 17 kDa prenyl binding protein (PBP), initially believed to be the fourth (δ) subunit of rod PDE6, was also used to solubilize membrane-associated PDE6. Further purification of GARP2 to apparent homogeneity was carried out by size exclusion chromatography on Superdex 200. Recombinant PBP and GARP2 were expressed and purified using standard protocols. Results: GARP2 is tightly associated with membrane-bound rod PDE6 upon hypotonic release of PDE6 from ROS membranes. Size exclusion chromatography indicates an apparent molecular weight >250 kDa for this complex. GARP2 co-purifies with PDE6 upon ion-exchange, hydroxyapatite, and size exclusion chromatography. To dissociate the GARP2-PDE6 complex, high salt treatment and HIC was used to prepare GARP2 depleted of 95% of the PDE6. An alternative purification strategy employed the 17 kDa PBP to solubilize PDE6 from ROS membranes. In addition to PDE6, PBP solubilized GARP1, but not GARP2. GARP2 was subsequently eluted from the membranes with a hypotonic buffer, and purified to apparent homogeneity by size exclusion chromatography. The specificity of PBP to disrupt GARP2-PDE6 interactions was confirmed by addition of PBP to the partially purified, soluble GARP2-PDE6 complex, and observation of complete dissociation of GARP2 from PDE6. Illumination of ROS membranes differentially affects GARP2 and PDE6 compartmentation. While 70-80% of PDE6 migrates to a detergent-insoluble membrane fraction upon illumination [Seno et al., JBC 276, 20813 (2001)], GARP2 is found only in the detergent-soluble fraction of both dark-adapted and illuminated ROS membranes. The ability of purified GARP2 to alter the catalytic and regulatory properties of PDE6 is currently under investigation. Conclusions: GARP2 forms a high-affinity interaction with rod PDE6 that can be disrupted by either illumination of ROS membranes or addition of exogenous PBP. The role of this reversible interaction between GARP2 and PDE6 is unknown, but could represent a mechanism to desensitize rod PDE6 activation under light-adapted conditions.
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