Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

Peshenko, Igor V.; Dizhoor, Alexander M.

doi:10.1074/jbc.c400065200

Cited by 88 publications

(93 citation statements)

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“…Recombinant myristoylated bovine and mouse GCAP-2, GCAP-1, and neurocalcin were expressed from pET11d vector (Novagen/EMD) in a BLR(DE3)pLysS Escherichia coli strain harboring yeast N-myristoyltransferase and purified as described previously in full detail (12,16,17).…”

Section: Methodsmentioning

confidence: 99%

“…RetGC Assay-All experiments were conducted under infrared light as described (12). The assay mixture (25 l) contained 30 mM MOPS/ KOH, pH 7.2, 60 mM KCl, 5 mM NaCl, 1 mM dithiothreitol, 2 mM Ca 2ϩ /EGTA buffer, 1 mM free Mg 2ϩ , zaprinast and dipyridamole (25 M each), leupeptin and aprotinin (10 g/ml each), 0.3 mM ATP, 4 mM cGMP, 1 mM GTP, ϳ1 Ci of [␣-…”

Section: Methodsmentioning

confidence: 99%

“…Photoreceptor outer segment membranes were isolated under infrared illumination from frozen dark-adapted bovine retinas (Lawson and Lawson) using sucrose density gradient centrifugation and were washed in low salt buffer to remove endogenous GCAPs as described previously (12,16). Soluble Retinal Extract (S 100 )-Fresh bovine eyes obtained at a local slaughterhouse were dark-adapted for 2 h on ice, dissected under infrared light, and homogenized in 0.5 ml of buffer A (5 mM sodium phosphate buffer, pH 7.2, 50 mM NaCl) per retina on ice by 10 strokes in a Dounce homogenizer.…”

Section: Methodsmentioning

confidence: 99%

“…The recoverin-like neuronal calcium sensor proteins have four EF-hand-type Ca 2ϩ -binding domains and an Nterminal Gly residue acylated with fatty acid residues. Only three of the EF-hand structures in GCAPs are true Ca 2ϩ -binding domains in which Ca 2ϩ sensitivity is adjusted to the range within which the intracellular free Ca 2ϩ varies between light and dark by intracellular free Mg 2ϩ (12). The N-terminal EF-hand-related motif EF-1 cannot bind Ca 2ϩ but strongly contributes to the GCAP interaction with retGC (13,14).…”

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confidence: 99%

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Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca 2؉ but activate retGC when Ca 2؉ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca 2؉ . We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotidedependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser 201 , had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca 2؉ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca 2؉ -insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca 2؉ -sensitive manner. The Ca 2؉ -dependent conformational changes in GCAP-2 affect the areas around Glu 62 residue in the entering helix of EFhand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu 136 -Glu 138 between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser 201 from the constraint caused by the Ca 2؉ -bound conformation.Photoisomerized rhodopsin triggers hydrolysis of cGMP through the activation of the G-protein transducin, which stimulates cGMP phosphodiesterase and thus causes cGMPgated channels to close (see Refs. 1-3 for review). Rods and cones rapidly recover to their resting potential after excitation induced by a brief non-saturating exposure to light. They also adapt to a constant background illumination by decreasing the amplitude and accelerating the kinetics of their electrical responses to a standard test flash (see Refs. 1-3 for review). Reopening of the cGMP-gated channels during recovery and light adaptation requires acceleration of cGMP synthesis by guanylyl cyclase, a process controlled by the level of intracellular free Ca 2ϩ . In the dark, Ca 2ϩ extruded from the photoreceptor outer segment by a Na ϩ /K ϩ ,Ca 2ϩ exchanger re-enters the outer segment through the open cGMP-gated channels so that the free intracellular outer segment Ca 2ϩ concentrations reach 250 nM in mammals and 350 -450 nM in lower vertebrates (4 -6). In the light, cGMP is hydrolyzed, and these channels close while the exchanger continues to extrude Ca 2ϩ ions from the photoreceptor outer segment; therefore, Ca 2ϩ concentrations decrease nearly 10-fold (4 -7). Ca 2ϩ -binding proteins GCAP-1 1 and GCAP-2 activate retGC1 and retGC2 (and their homologs found in photoreceptors of different vertebr...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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“…Magnesium is not normally considered a regulator, but recent in vivo measurements have detected changes in free Mg 2ϩ concentrations in cortical neurons after treatment with neurotransmitter (25). Other NCS proteins such as GCAPs, VILIP, and NCS-1 also bind Mg 2ϩ and exhibit Mg 2ϩ -induced effects (26,27 (10). The recombinant DREAM-C protein was expressed in soluble form and could be purified in milligram amounts, in contrast to the recombinant full-length protein, which appeared insoluble in bacterial extracts.…”

Section: Dreammentioning

confidence: 99%

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

Osawa

Dace

Tong

et al. 2005

Journal of Biological Chemistry

106

156

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The myristoylation of guanylate cyclase‐activating protein‐2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

2011

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Guanylate cyclase activating protein-2 (GCAP-2) is a Ca 21 -binding protein of the neuronal calcium sensor (NCS) family. Ca 21 -free GCAP-2 activates the retinal rod outer segment guanylate cyclases ROS-GC1 and 2. Native GCAP-2 is N-terminally myristoylated. Detailed structural information on the Ca 21 -dependent conformational switch of GCAP-2 is missing so far, as no atomic resolution structures of the Ca 21 -free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca 21 -myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP-1, a Ca 21 -independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non-myristoylated GCAP-2 in their Ca 21 -bound and Ca 21 -free forms, respectively, to gain information on the nature of the Ca 21 -dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca 21 , the stability of the myristoylated and non-myristoylated forms was indistinguishable. Ca 21 exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP-2. The stability data were corroborated by dye binding experiments performed to probe the solvent-accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent-exposed in Ca 21 -free GCAP-2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca 21 -bound state.

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Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

Cited by 88 publications

References 31 publications

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

The myristoylation of guanylate cyclase‐activating protein‐2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

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Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

Cited by 88 publications

References 31 publications

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

The myristoylation of guanylate cyclase‐activating protein‐2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca2+

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The myristoylation of guanylate cyclase‐activating protein‐2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺