Figure 8.

GCAP can displace RD3 from RetGC1.A, inhibition of RetGC1 activity by RD3 in vitro. The RetGC1 activity in HEK293 membranes activated by 1.5 μM wildtype Mg²⁺ GCAP1 (●, ◯) or Mg²⁺ GCAP1^L176F (▲, △) was assayed in the presence of increasing concentrations of wildtype RD3 (•, ▲) or RD3^4ET (◯, △). B, cosedimentation assay of RD3^4ET binding to RetGC1 using ultracentrifugation. Immunoblots of HEK293 membrane pellets, either containing (+) or not containing (−) expressed RetGC1, were obtained by ultracentrifugation after preincubation with 50 nM RD3^4ET in the presence of indicated concentrations of L176F GCAP1. The immunoblot was probed by anti-RetGC1 (top) or anti-RD3 (bottom) antibody as described in the Experimental procedures section. GCAP, guanylyl cyclase–activating protein; HEK293, human embryonic kidney 293 cells; RD3, retinal degeneration-3 protein; RetGC, the retinal membrane guanylyl cyclase.