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.
Highlights
Calcium is the major regulator of the physiological responses in photoreceptor cells
Recombinant Guanylyl cyclase activating proteins (GCAPs)-1 and Its Mutants—All mutations were of retinal guanylyl cyclase (RetGC)—In our previous study we found that disabling all incorporated into bovine GCAP-1 cDNA by PCR using a “splic- three metal-binding EF-hands in GCAP-1 by mutations that ing by overlap extension” technique [27]
Free myristic acid was added from a concentrated ethanol solution to the suspension of bacterial cells to a final concentration of make GCAP-1 a constitutive activator of RetGC it should be made unable to bind Ca2ϩ, yet still retain the ability to bind Mg2ϩ in its EF-hand(s)
Summary
Calcium is the major regulator of the physiological responses in photoreceptor cells. There are multiple reports that substitutions of the first and last coordinating amino acids inactivate EF-hands in NCS proteins [17, 22,23,24,25] Among these are the observations that inactivation of all three metal-binding EF-hands in GCAP-2 by substitution of the last Glu in EF-hands 2 and 3 with Gln and the first Asp in the EF-hand 4 with Asn make GCAP-2 a constitutive, insensitive to Ca2ϩ activator of RetGC [24]. We contained 30 mM MOPS/KOH (pH 7.2), 60 mM KCl, 4 mM concluded from the previous study [22] that, because cation NaCl, 1 mM dithiothreitol, 5 mM free Mg2ϩ, 2 mM Ca/EGTA binding was required for inhibition of RetGC, but was buffer, 0.3 mM ATP, 4 mM cGMP, 1 mM GTP, 1 Ci of critical for RetGC activation under physiological condi- [␣-32P]GTP, 0.1 Ci of [8-3H]cGMP, GCAP-1, and HEK 293 tions, the general view on the functioning of GCAPs as metal- cell membranes. The functions of the specific EF-hands in aliquots were analyzed by TLC using fluorescent plastic-backed
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