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PV Lishko, KA Martemyanov, JA Hopp, VY Arshavsky; Specific Binding of RGS9-Gß5 to Photoreceptor Membranes Enhances its GAP Activity, Requires a Protein Anchor and Occurs Via the DEP Domain . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2896.
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Purpose: The inactivation of transducin during recovery from the photoresponse requires the hydrolysis of transducin-bound GTP. In photoreceptors this reaction is catalyzed by the GTPase activating protein (GAP) RGS9 which exists as a complex with type 5 G protein ß subunit (Gß5). RGS9-Gß5 has been shown tightly associated with disc membranes. Our goals were: 1) to elucidate whether RGS9-Gß5 binding to photoreceptor membranes requires the presence of a membrane-associated protein anchor; 2) to test whether this binding regulates the GAP activity of RGS9-Gß5; 3) to identify the epitope of RGS9-Gß5 responsible for the membrane attachment. Methods: Recombinant RGS9-Gß5 and an array of its deletion mutants were expressed in the Sf9 baculovirus system. Their ability to interact with photoreceptor membranes and catalytic activity were studied by standard techniques. Results: We found that photoreceptor membranes contain a limited number of high affinity sites for RGS9-Gß5 binding. These sites are normally occupied by endogenous RGS9-Gß5 and are not available for the recombinant protein. However, they become available after treating the photoreceptor membranes with urea or high pH buffer. These sites disappear upon treatments with various proteases indicating that they are provided by a membrane-associated protein. The GAP activity of RGS9-Gß5 bound to these sites is higher than the activity of soluble protein by over an order of magnitude. The analysis of RGS9-Gß5 deletion mutants indicates that the binding requires the presence of both DEP and catalytic domains of RGS9 and does not require the presence of the C-terminus of RGS9, as was recently reported (He et. al., J. Biol. Chem., in press). Conclusion: Our data indicate that photoreceptors contain a specific protein that anchors RGS9-Gß5 to the surface of the disc membranes. This interaction results in a significant increase in the ability of RGS9-Gß5 to activate transducin GTPase and requires both the DEP and catalytic domains of RGS9-Gß5.
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