RESUMO
Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.
Assuntos
Sinalização do Cálcio/genética , Receptor Quinase 1 Acoplada a Proteína G/genética , Neoplasias/genética , Proteínas Sensoras de Cálcio Neuronal/genética , Neurônios/metabolismo , Neuropeptídeos/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Dimerização , Dissulfetos/química , Motivos EF Hand/genética , Células HEK293 , Humanos , Cinética , Neoplasias/patologia , Proteínas Sensoras de Cálcio Neuronal/antagonistas & inibidores , Neurônios/química , Neuropeptídeos/antagonistas & inibidores , Oxirredução , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Zinco/metabolismoRESUMO
The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson's disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.
Assuntos
Cálcio/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/genética , Calmodulina/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Sensoras de Cálcio Neuronal/antagonistas & inibidores , Proteínas Sensoras de Cálcio Neuronal/genética , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
There are two major forms of long-term depression (LTD) of synaptic transmission in the central nervous system that require activation of either N-methyl-D-aspartate receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). In synapses in the perirhinal cortex, we have directly compared the Ca(2+) signaling mechanisms involved in NMDAR-LTD and mGluR-LTD. While both forms of LTD involve Ca(2+) release from intracellular stores, the Ca(2+) sensors involved are different; NMDAR-LTD involves calmodulin, while mGluR-LTD involves the neuronal Ca(2+) sensor (NCS) protein NCS-1. In addition, there is a specific requirement for IP3 and PKC, as well as protein interacting with C kinase (PICK-1) in mGluR-LTD. NCS-1 binds directly to PICK1 via its BAR domain in a Ca(2+)-dependent manner. Furthermore, the NCS-1-PICK1 association is stimulated by activation of mGluRs, but not NMDARs, and introduction of a PICK1 BAR domain fusion protein specifically blocks mGluR-LTD. Thus, NCS-1 plays a distinct role in mGluR-LTD.