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1.
Biochim Biophys Acta ; 1854(10 Pt A): 1325-37, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26001899

RESUMO

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.


Assuntos
Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Guanilato Ciclase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Recoverina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Sinalização do Cálcio , Bovinos , Receptor Quinase 1 Acoplada a Proteína G/genética , Regulação da Expressão Gênica , Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/química , Proteínas Ativadoras de Guanilato Ciclase/genética , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Recoverina/química , Recoverina/genética , Alinhamento de Sequência
2.
Front Mol Neurosci ; 5: 28, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22408603

RESUMO

Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca(2+)/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca(2+)-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca(2+)-sensing protein. The synergetic inhibition of RK by both Ca(2+)-sensors occurs over a broader range of Ca(2+)-concentration than by recoverin alone, indicating increased Ca(2+)-sensitivity of RK regulation in the presence of both Ca(2+)-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.

3.
Biochem J ; 435(2): 441-50, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21299498

RESUMO

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.


Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Recoverina/química , Recoverina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Animais , Sítios de Ligação/genética , Bovinos , Receptor Quinase 1 Acoplada a Proteína G/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Recoverina/genética , Homologia de Sequência de Aminoácidos
4.
J Neurochem ; 110(1): 72-9, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19457073

RESUMO

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca(2+)] in the dark state of the photoreceptor cell. Decreasing [Ca(2+)] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca(2+)-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca(2+)-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.


Assuntos
Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Recoverina/metabolismo , Rodopsina/metabolismo , Visão Ocular/fisiologia , Regulação Alostérica/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Domínio Catalítico/fisiologia , Bovinos , Receptor Quinase 1 Acoplada a Proteína G/química , Ligação Proteica/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína/fisiologia , Recoverina/química , Rodopsina/química
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