Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins

Abstract

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.

Fig. 1

RD3 inhibits RetGC activity at submicromolar concentrations. A. Wild type mouse ROS were titrated with human recombinant RD3 in the presence of 1 mM free Mg²⁺ and 2 mM EGTA. Inset, Coomassie Brilliant Blue R250 - stained gel of human RD3 isolated from E. coli (left lane) and molecular weight standards (right lane). B. Human recombinant RetGC1 activated by 1.5 μM GCAP1 in the presence of 1 mM free Mg²⁺ and 2 mM EGTA was assayed at different concentrations of the human recombinant RD3 expressed and purified from E. coli. C. Human recombinant RetGC1 was activated by 1.5 μM bovine GCAP1 in the presence of 1 mM free Mg²⁺ and 2 mM EGTA and titrated with protein extracts from HEK293 cells either expressing or not expressing RD3. To exclude the possibility of a non-specific effect of different total protein concentrations, the total amount of protein was equilibrated with a control protein extract in every assay mixture. Inset, immunoblotting of HEK293 cell extracts probed with anti-RD3 antibody 497, left lane - non-transfected cells; right lane – RD3 plasmid-transfected cells (notice that there is no endogenous RD3 expression in the non-transfected cells). The data in A–D are fitted by the equation, a = (a_max − a_min)/(1+[RD3]/IC₅₀) + a_min; where a_max and a_min are the maximal and minimal activity of guanylyl cyclase in the experiment, respectively, and the IC₅₀ is the concentration of RD3 producing 50% inhibition. D–F. The effect of RD3 on guanylyl cyclase catalytic activity in ROS fractions measured in the absence of GCAPs. The RetGC activity in ROS fraction from GCAP1,2^−/− mouse retinas (24) was titrated with the purified E. coli-expressed RD3 in the presence of saturating 10 mM Mg²⁺ and 2 mM EGTA; GTP concentration in D was 1 mM and in E–F varied as indicated. E. Michaelis plot of the non-stimulated RetGC catalytic activity in the absence (●) or in the presence of 30 nM (■) or 60 nM (▲) purified recombinant RD3, representative from three similar independent experiments. F. Lineweaver-Burke plot for data from panel E illustrates a respective ~2.5-fold and 5-fold suppression of the V_max by 30 nM and 60 nM RD3 without a major effect on K_mGTP. The K_i calculated using the equation for a noncompetitive inhibition, V_i=V_max(1+[RD3]/K_i)⁻¹, from three independent experiments was 19 nM ± 7 SD. The activity in assays containing retinal membranes is presented per rhodopsin content, in membranes expressing recombinant RetGC it is normalized by the maximal activity for each series of the membrane preparations.

Fig. 2

RD3 inhibits RetGC activation through competition with GCAPs. A. The guanylyl cyclase activity in HEK293 homogenates containing RetGC1 expressed alone (●) or co-expressed with human RD3 (▲) was assayed in the presence of added recombinant GCAP1. The data were fitted assuming Michaelis hyperbolic function, a=a_max[GCAP]/(K_1/2+(GCAP)); equalized by RetGC1 content in both samples. B. Immunoblotting. The cells expressing RetGC1, either alone or co-transfected with RD3, from panel A were probed with anti-RetGC1 (left) or anti-RD3 polyclonal antibody 497 (right). Non-transfected HEK293 cells (leftmost in each panel) were used as a specificity control. C–F. Competition of RD3 with GCAP1 and GCAP2 in RetGC assay. C, D. RetGC1 expressed in HEK293 cells was activated by purified GCAP1 (C) or GCAP2 (D) in the absence (●) or in the presence of 3 nM RD3 (▲) or 9 nM RD3 (■). The data were fitted by Michaelis hyperbolic function. Maximal RetGC1 activity (a_max, mean ± SD) at 0 nm, 3 nM or 9 nM RD3 was 4.8 ± 0.13, 4.8 ± 0.11, and 4.6 ± 0.16 nmol/min/mg protein, respectively, when activated by GCAP1 and 2.1 ± 0.05, 2.6 ± 0.2 and 2.7 ± 0.1 nmol/min/mg when activated by GCAP2. The respective concentrations of GCAP producing half-maximal activation (K_1/2) were 1.1 ± 0.12, 4.1 ± 0.6, and 7.5 ± 0.5 μM (GCAP1) and 5.9 ± 0.6, 19 ± 2.6, and 36 ± 1.5 μM (GCAP2). E, F. Double-reciprocal plots related to panels C and D, respectively.

Fig. 3

RetGC activation by GCAP suppressed by RD3 retains near normal sensitivity to Ca²⁺and Mg²⁺. A–C. ROS fraction isolated from C57B6 mouse retinas was assayed for RetGC activity (open symbols) at various free Ca²⁺ concentrations and either 1 mM (A) or 6 mM (B) free Mg²⁺ concentrations in the presence or in the absence of 0.1 μM or 0.5 μM recombinant RD3 as indicated; in panel C, the activities normalized to the maximal RetGC activity in each assay series are shown as a function of free Ca²⁺ concentrations. D–F. Membrane fraction from HEK 293 cells expressing human RetGC1 (filled symbols) was reconstituted with 5 μM recombinant GCAP1 in the absence or in the presence of 10 nM and 30 nM RD3 as indicated and assayed as described under Experimental Procedures.

Fig. 4

Mutations found in human patients with retinal diseases (15) affect RD3 expression in HEK293 cells. A. Expression of RD3 mutants in HEK293 cells. Human wild type and mutant RD3 tagged with the 1D4 peptide at the C-terminus were detected by immunoblotting as described in Experimental Procedures. Actin immunostaining (upper panel) was used as a protein load control. B. The average band intensities of the mutant RD3 proteins relative to WT RD3 band were determined from 3 independent experiments described in panel A (mean ± SD). C. Co-IP experiment. RetGC1 and RD3 were co-expressed as described in Experimental procedures and co-IP using Sepharose-coupled anti-RD3 9D12 antibody. The samples applied to the column (marked “I” for “input”) and the eluted fractions (marked “B” for “bound”) were analyzed by immunoblotting probed with anti-RetGC1 (GC-8A5; upper panel) and anti-RD3 (RD3-9D12; lower panel) antibodies. The G57V RD3 mutant that fails to express in HEK293 cells was used as a control to confirm that RetGC1 does not bind nonspecifically to the 9D12 antibody-coupled Sepharose. The lower band on the blot is specific for RD3-transfected cells and is most likely a partially proteolyzed RD3 polypeptide.

Fig. 5

Effect of mutations in RD3 found in human patients with retinal diseases on RetGC activity in vitro. A. Wild type and mutant RD3 (15) expressed in E. coli were stained by Coomassie Brilliant Blue R-250 after SDS PAGE, 15% gel. For the use in subsequent RetGC assays, RD3 concentration in each case was that of the main band. B. Inhibition of RetGC1 activity by the E. coli - expressed RD3 mutants. RetGC activity in independent assays was normalized per activity measured in the absence of RD3. Recombinant RetGC1 expressed in HEK293 cells was reconstituted with 1.5 μM GCAP1 at indicated concentrations of RD3 (○) or its mutants: W6R/E23D (●), G35R (△), G57V (□), R68W (■), K130M (▲), and F100ter (◆). The IC₅₀ values (mean ± SE, n) for the inhibition were 4.6 ± 1.3 nM, 5 (WT); 6.4 ± 2 nM, 3 (W6R/E23D); 7.9 ± 1 nM, 3 (G35R); 68 ± 13 nM, 3 (G57V); 17.3 ± 3.8 nM, 3 (R68W); and 14.3 ± 2.4 nM, 3 (K130M); the IC₅₀ for the F100ter was ≫10 μM. The difference in the IC₅₀ values from the wild type was significant (p in unpaired Student t-test assuming equal variance) for the G57V (<0.001), F100ter (<0.0001), R68W (0.012), and K130M (0.014) mutants.

Fig. 6

A diagram depicting the inhibition of RetGC by RD3. A RetGC homodimer forms an active site by combining the Mg²⁺-coordinating center from one subunit with the GTP binding center from another subunit (–29). There are two symmetrical active sites in the dimer, but only one such site is depicted here for simplicity. The low RetGC basal catalytic activity (left) becomes accelerated up to 20–100 fold (21, 31) when GCAPs bind the cytoplasmic portion of RetGC (middle). RD3 binding to the cyclase (right) inhibits the RetGC catalytic activity, but does not prevent binding of the Mg²⁺GTP substrate in the active site; at the same time, RD3 acts as a negative high-affinity modulator of the RetGC/GCAP complex by displacing GCAP – this prevents dynamic activation by GCAPs via Ca²⁺-feedback mechanism. The GCAP-stimulated activity is much higher than the basal RetGC activity (21, 31); therefore, the strongest effect of RD3 as a negative allosteric modulator of the cyclase results from its competition with GCAP. Other explanations are provided in the Discussion.