Peripheral association of RGS9 with rod outer segment membranes. RGS9 immunoblots are shown for ROS membranes subjected to various washing procedures, as described in the text. Lanes were loaded with equivalent fractions of pellets (p) or supernatants (s). (A) Sequential washes with hypertonic buffer (Hyper); hypotonic buffer (Hypo); 4 M urea (Urea); GAPN moderate ionic strength buffer (GAPN). (B) Treatment with 100 mM sodium carbonate, pH 12.0. (C) Treatment with 1 M hydroxylamine.

Removal of ROS GAP activity by immunodepletion of RGS9. (A) RGS9 antibodies were immobilized on protein A-Sepharose and incubated with OG-solubilized ROS as described in the text. After removal by centrifugation, the ROS supernatants were analyzed for ROS GAP activity, PDEγ-enhanced ROS GAP activity, and RGS9 protein remaining in the supernatant (immunoblot, inset). Control samples are: 1, solubilized ROS extract; 2, solubilized extract after incubation with immobilized pre-immune rabbit antibodies; and 3, solubilized extract after incubation with RGS9 antibodies, which had been pre-incubated with recombinant RGS9 bound to Ni²⁺-NTA agarose prior to immobilization on protein A-Sepharose. Depleted extract, solubilized ROS extract after incubation with RGS9 antibodies, which had been pre-incubated with Ni²⁺-NTA agarose only prior to immobilization on protein A-agarose. For assays containing PDEγ (hatched boxes), a final concentration of 1.84 μM was used. (B) Immunodepletion by directly coupled antibodies. Either CNBr-activated Sepharose 4B with covalently coupled purified RGS9 antibodies or rabbit IgG-agarose was incubated with OG-solubilized ROS (control 4), and the supernatants were assayed for PDEγ-enhanced ROS GAP activity. PDEγ (hatched boxes) was present at 1.17 μM. (C) Extract was immunodepleted as in A, without preincubation of IgG with Ni²⁺-NTA agarose, and recombinant RGS9d (6) was added (10 μM) with or without PDEγ (0.3 μM). Control #1 is as in A.

Solubilization of RGS9 and ROS GAP activity by octyl glucoside. (A) Immunoblot of supernatants from ROS membranes washed with the indicated concentrations (mM) of octyl glucoside. (B) GTP hydrolysis acceleration induced by supernatants of ROS membranes washed with indicated concentrations of octyl glucoside. GTP hydrolysis of transducin was measured under single-turnover conditions in the presence of 920 nM PDEγ.

Immunolocalization of RGS9 in bovine retina by monoclonal antibody. (A) RGS9 immunoblot showing labeling of RGS9 from bovine ROS and full-length and truncated forms of bacterially expressed RGS9. ROS, bovine ROS (20 μg rhodopsin). Lane 1, His-RGS9 (delta30N, 0.6 μg) corresponding to aa 31–484. Lane 2, His-RGS9c (0.6 μg) corresponding to aa 226–484. Lane 3, His-RGS9d (0.6 μg) corresponding to aa 291–418. (B–D) Confocal immunolocalization. RGS9 immunolabeling (B and C) is present in rod and cone photoreceptors. These sensory neurons extend from the outer segment layer (OS) to the outer plexiform layer (OPL) where their axons terminate. Antibody labeling is more prominent in the cones, whose cell soma are adjacent to the IS layer, in the outer most tier of the outer nuclear layer (ONL). Antibody labeling was not observed in the inner nuclear layer (INL), inner plexiform layer (IPL), or ganglion cell layer (GCL). At higher magnification (C), the subcellular distribution of RGS9 in rods and cones is demonstrated. Cone OS are heavily labeled (arrowheads), whereas rod OS (}) are less intensely labeled. Rod and cone IS are also RGS9 immunopositive, although labeling is more pronounced in the cones. Cone photoreceptors terminate in the OPL where their axons form synaptic terminals. Cone axons and synaptic terminals (arrow) are positive for RGS9. (D) RGS9 immunolabeling is abolished by preadsorbing the antibody with purified RGS9 protein coupled to CNBr-activated Sepharose. Scale bars are 50 μm in B and D and 20 μm in C.