Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Abstract

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.

Fig. 1. EF-hands of bovine GCAP-1 and mutations used in this study

Mutations introduced in EF-hands of GCAP-1: D64N, D100N/D102G, and D144N/D148G to disable both Ca²⁺ and Mg²⁺ binding to EF-hand 2, 3 and 4, respectively; E75Q, E111Q and E155Q to disable only Ca²⁺-binding to EF-hand 2, 3 and 4, respectively.

Fig. 2. The Ca²⁺ sensitivity of RetGC-1 regulation by wild type GCAP-1 and its EF(2,3,4)⁻ mutants

Recombinant RetGC-1 was assayed at various free Ca²⁺ concentrations in the presence of 5 µM wild type GCAP-1 (○), E75Q/E111Q/E155Q GCAP-1 (■), or D64N/D100N/D102G/D144N/D148G GCAP-1(▲), at saturating 5 mM free Mg²⁺. The data for wild type GCAP-1 were fitted by the equation, A = (A_max−A_min)/(1 + ([Ca]_f/[Ca]_1/2)ⁿ) + A_min, where A is the activity of RetGC-1, A_max and A_min is the maximal and minimal activity of RetGC in the assay, respectively, [Ca]_1/2 is the free Ca²⁺ concentration required for half-maximal inhibition of RetGC-1 by GCAP, n is the cooperativity coefficient. For other conditions of the assay see EXPERIMENTAL PROCEDURES.

Fig. 3. The effect of various substitutions in GCAP-1 EF-hands on RetGC-1 activation

Mutations that either prevented or preserved Mg²⁺ binding were introduced in the individual EF-hands of GCAP-1 and the activity of RetGC-1 was measured in the presence of 1 mM EGTA, 5 mM free Mg²⁺, and increasing concentrations of GCAP-1 as described in EXPERIMENTAL PROCEDURES. A, Comparison of EF(2)⁻ mutants: (○), wild type; (●), E75Q; (▲), D64N B, Comparison of EF(3)⁻ mutants: (○), wild type; (■), E111Q; (▼), D100N/D102G C, Comparison of EF(4)⁻ mutants: (○), wild type; (□), D144N/D148G; (△), E155Q. D, Comparison of EF(2,3)⁻ mutants: (○), wild type; (■), E75Q/E111Q; (●), D64N/D100N/D102G. E, Competition between wild type GCAP-1 and its EF(2,3)⁻ mutant, D64N/D100N/D102G. Increasing concentrations of the D64N/D100N/D102G mutant were added into GC assay mixture, alone (○) or in the presence of 3 µM wild type GCAP-1 (●). The assay mixture contained 1 mM EGTA and 5 mM free Mg²⁺. The data were fitted by the equation, A = A_max [GCAP]/(K_1/2 + [GCAP]), where A is the activity of RetGC-1 in the assay, A_max is the maximal activity of RetGC, [GCAP] is the concentration of GCAP, K_1/2 is the concentration of GCAP required for half-maximal activation of RetGC-1. The values of A_max and K_1/2 are summarized in Table 1.

Fig. 4. Effect of disabling of Mg²⁺-binding in EF-hand 2 on the single Trp21 fluorescence in EF-hand 1

Mutations, E75Q (▲), or D64N (●), were introduced to inactivate EF-hand 2 in a GCAP-1 mutant, W51F/W94F (□), that has a single remaining Trp21 residue (22). The fluorescence of the Trp21 was recorded as a function of Mg²⁺ in the presence of 1 mM EGTA.

Fig. 5. Effect of EF-hand 2 transition between its Mg²⁺- and Ca²⁺-bound state on RetGC-1 activation

A, Recombinant RetGC-1 was assayed for GC activity at 5 mM free Mg²⁺ and variable free Ca²⁺ concentrations in the presence of 2 µM GCAP-1: (○), wild type; (●), D100N/D102G/D144N/D148G; (□), E75Q/D100N/D102G/D144N/D148G. B, C, Dose-dependence of RetGC-1 activation by GCAP-1(D100N/D102G/D144N/D148G) (B) or GCAP-1(E75Q/D100N/D102G/D144N/D148G) (C) in the presence of 1 mM EGTA (open symbols) or 10 µM free Ca²⁺ (filled symbols). The concentration of free Mg²⁺ in the GC assay was 5 mM. The data were fitted as described in the legend to Fig. 3. The values of A_max and K_1/2 are summarized in Table 2.

Fig. 6. Effect of individual EF-hands inactivation in GCAP-1 on Ca²⁺ sensitivity of RetGC-1 regulation

RetGC activity was measured as a function of free Ca²⁺ concentrations at 5 mM free Mg²⁺ in the presence of 2 µM wild type GCAP-1, (○); E75Q, (●); E111Q, (■); E155Q, (□); D144N/D148G, (▼) or E111Q/D144N/D148G (△). The data for wild type GCAP-1 and E75Q mutant were fitted by the equation, A = (A_max−A_min)/(1 + ([Ca]_f/[Ca]_1/2)ⁿ) + A_min, where A is the activity of RetGC-1, A_max and A_min is the maximal and minimal activity of RetGC in the assay, respectively, [Ca]_1/2 is the free Ca²⁺ concentration required for half-maximal inhibition of RetGC-1 by GCAP, n is the Hill coefficient. For other conditions of the assay see EXPERIMENTAL PROCEDURES.

Fig. 7

A, In light-adapted conditions, all three metal-binding EF-hands in GCAP-1 are occupied by Mg²⁺, however, only Mg²⁺ binding in EF-hands 2 and 3 is required for RetGC stimulation. The apo forms of these EF-hands do not create the proper conformation for GCAP-1. In the dark, all three EF-hands of GCAP-1 are predominantly occupied by Ca²⁺, however, the main requirement for converting GCAP-1 into the “RetGC inhibitor” is binding of Ca²⁺ in EF-hand 4. Ca²⁺ binding in EF-hands 2 and 3 is primarily required for GCAP-1 to preserve binding to RetGC and to facilitate the high-affinity binding of Ca²⁺ to EF-hand 4. B. Ca²⁺/Mg²⁺ exchange in EF-hands of GCAP-1 between light and dark provides functional switch between its “RetGC activator” and “RetGC inhibitor” states. Apo form of GCAP-1 has no function. Other explanations are in DISCUSSION.