Mapping Sites in Guanylyl Cyclase Activating Protein-1 Required for Regulation of Photoreceptor Membrane Guanylyl Cyclases

Krylov, Dmitri M.; Niemi, Gregory A.; Dizhoor, Alexander M.; Hurley, James B.

doi:10.1074/jbc.274.16.10833

Cited by 55 publications

(54 citation statements)

References 24 publications

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“…In vitro, GCAP1 activates GC1 more efficiently than GC2 [170,171], whereas GCAP2 activates both GC1 and GC2 with similar efficiency [163,172,173]. In vivo, expression of GCAP1, the major form in cones, in GCAPs −/− retina restored normal response kinetics of cones as expected [174].…”

Section: +mentioning

confidence: 80%

Phototransduction in mouse rods and cones

Yau

2007

Pflugers Arch - Eur J Physiol

266

244

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Phototransduction is the process by which light triggers an electrical signal in a photoreceptor cell. Imageforming vision in vertebrates is mediated by two types of photoreceptors: the rods and the cones. In this review, we provide a summary of the success in which the mouse has served as a vertebrate model for studying rod phototransduction, with respect to both the activation and termination steps. Cones are still not as well-understood as rods partly because it is difficult to work with mouse cones due to their scarcity and fragility. The situation may change, however.

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Section: +mentioning

confidence: 80%

Phototransduction in mouse rods and cones

Yau

2007

Pflugers Arch - Eur J Physiol

266

244

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“…Wild-type GCAP-1 and its mutants used in this study also carried a D6S substitution that creates a recognition site for the yeast N-myristoyltransferase (26) and does not interfere with the RetGC regulation by the recombinant GCAP-1 (27,28). GCAP-1 cDNA was inserted into the NcoI/BamHI sites of the pET11d vector (Novagen/Calbiochem) and expressed under control of the isopropyl ␤-D-thiogalactopyranoside-regulated T7 promoter in a BL21(DE3)pLysS Escherichia coli strain (Novagen/Calbiochem) harboring a pBB131 plasmid for N-myristoyltransferase expression as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Ca2+ and Mg2+ Binding Properties of GCAP-1

Peshenko

Dizhoor

2006

Journal of Biological Chemistry

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102

108

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Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca 2؉ in the light and inhibits it in the dark when Ca 2؉ concentrations rise. We present the first direct evidence that Mg 2؉ -bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP- In this study, we continue to develop a concept for the processes through which Ca 2ϩ sensor proteins, GCAPs, 2 regulate their target enzyme, retinal guanylyl cyclase (RetGC), in photosensitive neurons of the retina between light and dark. In darkadapted photoreceptors, cGMP keeps a fraction of cGMPgated channels open, thus allowing Na ϩ and Ca 2ϩ to enter the outer segment (1, 2). Because Ca 2ϩ is continuously removed from the outer segments by a light-independent Na ϩ /K GCAPs are recoverin-like neuronal calcium sensor proteins within the EF-hand superfamily (14, 15). Like other recoverinlike proteins, GCAPs have four EF-hand domains, but based on their amino acid sequence only three of them are capable of binding metal ion. Although the general model of regulation requires that Ca 2ϩ be either bound or released by GCAPs, little is known about the actual Ca 2ϩ binding properties of individual GCAPs under physiologically relevant conditions or the actual conformational change in GCAPs that constitutes switching them between the activator and the inhibitor forms. A number of previous studies aimed to identify conformational changes in GCAP-1 between its activator and inhibitor forms using limited proteolysis (16 -18), chemical modification of endogenous cysteine residues (17), EPR spectroscopy (17), and tryptophan fluorescence spectroscopy (18 -21) were based on the assumption that GCAPs are only Ca 2ϩ -binding proteins and that the metalfree GCAPs are the activators of RetGC-1. Our recent finding demonstrated that Mg 2ϩ is essential for adjusting the Ca 2ϩ sensitivity of RetGC regulation by GCAPs to the actual physiological range of free Ca 2ϩ concentrations and provided the first evidence that GCAPs are both Ca 2ϩ and Mg 2ϩ sensor proteins (5, 13, 22) thus raising the possibility that at physiological free Ca 2ϩ and Mg 2ϩ concentrations in the light at least one of the EF-hands in GCAP-1 is in a Mg 2ϩ -bound, rather than metalfree, state. Importantly binding of Mg 2ϩ changes the intensity of the intrinsic tryptophan fluorescence of GCAP-1, and that implies that a significant structural difference exists between the Mg 2ϩ -bound and the metal-free GCAP-1 (13). In the present study, we determined both Ca 2ϩ and Mg 2ϩ binding properties of GCAP-1 using two independent methods and a wide range of mutations introduced in its EF-hand domains. We found the following. (i) At the physiological concentrations of free Mg 2ϩ and Ca 2ϩ that exist in the vertebrate photoreceptors in the light (3,23,24), three EF-hands in GCAP-1 are predominantly occupied with Mg 2ϩ ions; hence the physiological RetGC-activat...

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“…Site-directed Mutagenesis-All mutations were incorporated into bovine GCAP-2 cDNA by PCR using the splicing by overlap extension technique and Pfu DNA polymerase (Stratagene) as described previously (18,19 43,31,21,14, and 6 kDa. The position of GCAPs in the gel is indicated by the asterisk.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…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).The partial solution structure of the Ca 2ϩ -bound GCAP-2 established by Ames et al (15) resembles those of recoverin and neurocalcin and consists of two globular halves connected by a flexible "hinge" region. The NMR structure was established only for the Ca 2ϩ -loaded form of GCAP-2 and does not include the C-terminal portion of the protein; therefore, the exact conformational changes that occur when dissociation of the Ca 2ϩ ions causes GCAP-2 to transition into the activator conformation are not immediately apparent.…”

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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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Mapping Sites in Guanylyl Cyclase Activating Protein-1 Required for Regulation of Photoreceptor Membrane Guanylyl Cyclases

Cited by 55 publications

References 24 publications

Phototransduction in mouse rods and cones

Phototransduction in mouse rods and cones

Ca2+ and Mg2+ Binding Properties of GCAP-1

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

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