Conformational Changes in Guanylyl Cyclase-activating Protein 1 (GCAP1) and Its Tryptophan Mutants as a Function of Calcium Concentration

Sokal, Izabela; Otto‐Bruc, Annie; Surgucheva, Irina; Verlinde, Christophe L. M. J.; Wang, Chien Kao; Baehr, Wolfgang; Palczewski, Krzysztof

doi:10.1074/jbc.274.28.19829

Cited by 47 publications

(42 citation statements)

References 28 publications

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“…Bovine GCAP-1 exhibits Ca 2ϩ -sensitive changes in the intrinsic tryptophan fluorescence as a result of cation binding to its EF-hands (23)(24)(25). The fluorescence of myristoylated bovine GCAP-1 is at its maximum in the absence of Ca 2ϩ and Mg 2ϩ , and the changes in fluorescence as a function of free Ca 2ϩ ([Ca] f ) are biphasic (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

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

Peshenko

Dizhoor

2004

Journal of Biological Chemistry

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“…The subunits were dialyzed against measuring buffer (20 mM BTP, pH 7.4; 130 mM NaCl; 5 mM MgCl 2 ; 2 mM DTT), concentrated (Amicon YM-10 device), and stored at Ϫ40°C. Unphosphorylated rhodopsin kinase (RK) was purified as described previously (44), arrestin was purified from frozen darkadapted bovine retinas (18), GCAP1 was generated as described previously (53), recoverin was purified as described previously (21), and calmodulin was purchased from Calbiochem (catalog no. 208694).…”

Section: Methodsmentioning

confidence: 99%

Calcium-Dependent Assembly of Centrin-G-Protein Complex in Photoreceptor Cells

Pulvermüller

Gießl

Heck

et al. 2002

Molecular and Cellular Biology

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“…Because the Glu 62 residue located in the entering helix of EF-2 becomes exposed in Ca 2ϩ -free GCAP-2, the release of Ca 2ϩ most likely destabilizes the interaction between the entering helix of EF-2 and exiting helix of EF-3, potentially making the hinge region more accessible for external protein-protein interactions and/or reflecting a motion between the two globular parts of GCAP-2. It has also been speculated that such a motion takes place in GCAP-1 during binding and dissociation of Ca 2ϩ based on tryptophan fluorescence changes and tryptic digestion (45)(46)(47). Also, in Ca 2ϩ -free recoverin, the entering helix of EF-2 is solvent-exposed and structurally unstable.…”

Section: Ca 2ϩ -Dependent Conformational Changes In Gcap-2 50347mentioning

confidence: 99%

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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Conformational Changes in Guanylyl Cyclase-activating Protein 1 (GCAP1) and Its Tryptophan Mutants as a Function of Calcium Concentration

Abstract: Guanylyl cyclase-activating proteins (GCAPs

Cited by 47 publications

References 28 publications

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

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

Calcium-Dependent Assembly of Centrin-G-Protein Complex in Photoreceptor Cells

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

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