The effects of the
GUCY2D variants on guanylyl cyclase activity and regulation by GCAP1. (
A) Immunoblotting of the human
GUCY2D expressed in HEK293 cells. The HEK293 cells were transfected with
GUCY2D cDNA using calcium phosphate precipitation method and the membrane fractions from the transfected cultures were prepared as described in Methods. After the transfer from 7% SDS, the blot was probed by antibody against RetGC1 catalytic domain
53 using a Thermo Scientific SuperSignal West Femto Chemiluminescence Substrate. The amount of the
GUCY2D in subsequent biochemical analyses was normalized in all cases by the level of the chemiluminescence signal relative to the wild type. (
B) Dose dependence of
GUCY2D activation by GCAP1. The membrane fractions from HEK293 cells expressing wild-type, Y555S, L587V, R624Q, or G653E
GUCY2D were reconstituted with purified recombinant human GCAP1 (hGCAP1) and assayed at saturating 10-mM MgCl
2 in the presence of 2-mM EGTA. The data (mean ± SD) from three independent experiments were fitted (except for G653E, which had no detectable activity) using the function
A = (
Amax)/(1 + ([GCAP]
1/2/[GCAP])
h), where
A is the activity of the cyclase,
Amax is the maximal activity at saturation with GCAP, [GCAP]
1/2 is a GCAP concentration producing half-maximal stimulation, and
h is a Hill coefficient. The respective
Amax values (mean ± SD) from the fit for the wild-type, Y555S, L587V, and R624Q
GUCY2D were 39.4 ± 0.4, 6.1 ± 0.35, 4.6 ± 0.2, and 12.4 ± 0.7 nmol/min/mL (activity in all mutants was different from the wild type (
P <0.001,
t-test). The respective [GCAP]
1/2 values were 0.98 ± 0.03, 4.4 ± 0.43 (
P = 0.005), 2.4 ± 0.2 (
P = 0.005), and 4.1 ± 0.5 (
P = 0.01). (
C) The Ca
2+ sensitivity of GCAP-stimulated activation for wild-type, Y555S, L587V, and R624Q
GUCY2D. The cyclase activity in membranes containing recombinant
GUCY2D was assayed in the presence of 14-µM GCAP1 and Ca
2+/EGTA buffers, varying free Ca
2+ concentrations in the presence of 0.9-mM free Mg
2+ and normalized as a percentage of the maximal activity of each preparation. The data (mean ± SD,
n = 3) were fitted assuming a Hill function, such that
A% = 100/(1+([Ca]
f/[Ca]
1/2)
h), were
A% is the fractional activity of the cyclase, [Ca]
f is a free Ca
2+ concentration in the assay, [Ca]
1/2 is a free Ca
2+ concentration producing half-maximal inhibition of the cyclase activity, and
h is a Hill coefficient. The [Ca]
1/2 values for wild-type, Y555S, L587V, and R624Q
GUCY2D were 176 ± 6, 139 ± 8 (
P = 0.042), 114 ± 7 (
P < 0.001), and 57 ± 7 (
P < 0.001), respectively.