RESUMO
The control of calcium influx at the plasma membrane by endoplasmic reticulum (ER) calcium stores, a process common to invertebrates and vertebrates, is central to physiological calcium signalling and cellular calcium balance. Stromal interaction molecule 1 (STIM1) is a calcium sensor and regulatory protein localized to the ER. ORAI1 is a calcium channel in the plasma membrane (PM). In outline, STIM1 senses an ER-luminal calcium decrease, relocalizes to ER-PM junctions, and recruits and gates ORAI1 channels. Recent work, reviewed here, has offered detailed insight into the process of sensing and communicating ER calcium-store depletion, and particularly into the STIM1 conformational change that is the basis for communication between the ER and the PM.
Assuntos
Cálcio , Proteínas de Membrana , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteína ORAI1 , Molécula 1 de Interação Estromal/metabolismoRESUMO
STIM1 and STIM2 are endoplasmic reticulum (ER) membrane proteins that sense decreases in ER-luminal free Ca2+ and, through a conformational change in the STIM cytoplasmic domain, control gating of the plasma membrane Ca2+ channel ORAI1. To determine how STIM1 conveys a signal from the ER lumen to the cytoplasm, we studied the Ca2+-dependent conformational change of engineered STIM1 proteins in isolated ER membranes and, in parallel, physiological activation of these proteins in cells. We find that conserved "sentinel" features of the CC1 region help to prevent activation while Ca2+ is bound to STIM ER-luminal domains. Reduced ER-luminal Ca2+ drives a concerted conformational change, in which STIM luminal domains rearrange and the STIM transmembrane helices and initial parts of the CC1 regions pair in an extended coiled coil. This intradimer rearrangement overcomes the relatively weak CC1-SOAR/CAD interactions that hold STIM in an inactive conformation, releasing the SOAR/CAD domain to activate ORAI channels.
Assuntos
Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Ativação do Canal Iônico , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Transdução de Sinais , Molécula 1 de Interação Estromal/metabolismo , Citoplasma/genética , Retículo Endoplasmático/genética , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Molécula 1 de Interação Estromal/genéticaRESUMO
Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cisteína/metabolismo , Medição da Troca de Deutério , Fluorescência , Células HeLa , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Solubilidade , Relação Estrutura-AtividadeRESUMO
The ER-resident regulatory protein STIM1 triggers store-operated Ca(2+) entry by direct interaction with the plasma membrane Ca(2+) channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb(3+) luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Membrana Celular/metabolismo , Humanos , Proteína ORAI1 , Estrutura Secundária de Proteína , Molécula 1 de Interação EstromalRESUMO
The inducible T cell kinase (ITK) regulates type 2 (Th2) cytokines that provide defense against certain parasitic and bacterial infections and are involved in the pathogenesis of lung inflammation such as allergic asthma. Activation of ITK requires the interaction of its SH3 domain with the poly-proline region of its signaling partner, the SH2 domain containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). The specific disruption of the ITK-SH3/SLP-76 poly-proline interaction in vitro by a cell-permeable competitive inhibitor peptide (R9-QQP) interferes with the activation of ITK and the transduction of its cellular functions in T lymphocytes. In the present investigation, we assessed the effects of R9-QQP treatment on the induction of an in vivo immune response as represented by lung inflammation in a murine model of allergic asthma. We found that mice treated with R9-QQP and sensitized and challenged with the surrogate allergen ovalbumin (OVA) display significant inhibition of lung inflammation in a peptide-specific manner. Thus, parameters of the allergic response, such as airway hyper-responsiveness, suppression of inflammatory cell infiltration, reduction of bronchial mucus accumulation, and production of relevant cytokines from draining lymph nodes were significantly suppressed. These findings represent the first demonstration of the biological significance of the interaction between ITK and SLP-76 in the induction of an immune response in a whole animal model and specifically underscore the significance of the ITK-SH3 domain interaction with the poly-proline region of SLP-76 in the development of an inflammatory response. Furthermore, the experimental approach of intracellular peptide-mediated inhibition might be applicable to the study of other important intracellular interactions thus providing a paradigm for dissecting signal transduction pathways.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Imunidade/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Bronquíolos/efeitos dos fármacos , Bronquíolos/metabolismo , Bronquíolos/patologia , Peptídeos Penetradores de Células/química , Citocinas/biossíntese , Eosinofilia/complicações , Eosinofilia/imunologia , Eosinofilia/patologia , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Muco/metabolismo , Ovalbumina/imunologia , Pneumonia/complicações , Pneumonia/imunologia , Pneumonia/patologia , Proteínas Tirosina Quinases , Transdução de Sinais/efeitos dos fármacos , Células Th2/efeitos dos fármacosRESUMO
Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Conservada , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Interleucina-4/biossíntese , Células Jurkat , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Linfócitos T/metabolismo , TransfecçãoRESUMO
ITK-SH3-mediated interactions, both with exogenous ligands and via intermolecular self-association with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitution of the SH3 domain with that of the related kinase BTK (ITK-BTK((SH3))) was used to disrupt intermolecular self-association of ITK while maintaining canonical binding to exogenous ligands such as SLP-76. ITK-BTK((SH3)) displays reduced association with SLP-76 leading to inefficient transphosphorylation, reduced phosphorylation of PLCγ1, and diminished Th(2) cytokine production. In contrast, ITK-BTK((SH3)) displays no defect in its localization to the T-cell-APC contact site. Another mutation, Y511F, in the activation loop of ITK, impairs ITK activation. T cells expressing ITK-Y511F display defective phosphorylation of ITK and its downstream target PLCγ1, as well as significant inhibition of Th(2) cytokines. In contrast, the inducible localization of ITK-Y511F to the T cell-APC contact site and its association with SLP-76 are not affected. The presented data lend further support to the hypothesis that precise interactions between ITK and its signaling partners are required to support ITK signaling downstream of the TCR.