ABSTRACT
Using Baculovirus ............................................................................ 103 5.3 Reconstitution of Full-Length R9AP into Lipid Vesicles ........................... 105
5.3.1 Reconstitution of Puried R9AP into Vesicles Containing Phospholipids Only or Phospholipids and Rhodopsin ..................... 105
5.4 Single-Turnover GAP Assay for Puried Recombinant or Retinal Proteins ....................................................................................................... 106
5.5 Quantication of ROS Proteins Involved in GTPase Regulation by Quantitative Immunoblotting ...................................................................... 108
5.6 Immunoprecipitation of the GAP Complex ................................................ 111 5.7 Localization of Proteins in Rod Outer Segments by Subcellular
Fractionation and Immunouorescence ...................................................... 114 5.7.1 Gradient Copurication: A General Way to Determine Whether
a Protein Is Localized to ROS or a Contaminant from Other Parts of Retina.................................................................................. 114
5.7.2 Protein Localization by Immunouorescence ................................. 116 5.8 Phosphorylation of RGS9-1 ......................................................................... 117 References .............................................................................................................. 119
A major player in the regulation of timing and sensitivity of photoresponses is the GTPase accelerating protein, RGS9-1 (1-4). RGS9-1 binds to the α subunits of the rod and cone G-proteins, Gαt1 and Gαt2, when they are in their activated GTP-bound conformations, and speeds up the rates at which they hydrolyze GTP, thereupon returning to the inactive GDP-bound conformations. This conceptually simple function is complex in its biochemical details and regulation. In addition to RGS9-1, two other subunits, Gβ5L (5 –9) and R9AP (RGS9-1 anchor protein) (10-12), are required for the function and stability of the GTPase accelerating protein (GAP) complex, and the inhibitory subunit of the photoreceptor effector enzyme, PDE6γ, dramatically enhances the afnity of this complex for Gαt-GTP. The activity of the complex and the time resolution of vision depend on its concentration in the cells (13), and the concentrations are very different in rods and cones (1,14). The Gβ5 gene is subject to alternative splicing, and although only one splice variant, Gβ5L, is found in rods (15), both Gβ5L and Gβ5S are found associated with RGS9-1 in cones (14). RGS9-1 is also subject to Ca2+-regulated phosphorylation by protein kinase C (16,17), and may be further regulated by phosphoinositides. Levels of this complex have been shown to be essential for the timely recovery of photoresponse by loss-of-function studies in mice (18-21) and humans (22), and those proteins also control the rate-limiting step in vision conrmed by a recent discovery that overexpression of the whole complex speeds up the response (13). This chapter will focus on biochemical techniques used to characterize the molecular mechanisms of RGS9-1-GAP complex in phototransduction kinetics and their regulation.
