RESUMEN
Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina , Septinas , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Proteína ORAI1/genética , Molécula de Interacción Estromal 1RESUMEN
Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Actinas , Calcio , Citoesqueleto de Actina , Actinas/genética , Humanos , Mastocitos , Proteínas de Neoplasias/genética , Receptores de Cinasa C Activada/genética , TapsigarginaRESUMEN
Mammalian transient receptor potential (TRP) channels are major components of Ca2+ signaling pathways and control a diversity of physiological functions. Here, we report a specific role for TRPC1 in the entry of herpes simplex virus type 1 (HSV-1) into cells. HSV-1-induced Ca2+ release and entry were dependent on Orai1, STIM1, and TRPC1. Inhibition of Ca2+ entry or knockdown of these proteins attenuated viral entry and infection. HSV-1 glycoprotein D interacted with the third ectodomain of TRPC1, and this interaction facilitated viral entry. Knockout of TRPC1 attenuated HSV-1-induced ocular abnormality and morbidity in vivo in TRPC1-/- mice. There was a strong correlation between HSV-1 infection and plasma membrane localization of TRPC1 in epithelial cells within oral lesions in buccal biopsies from HSV-1-infected patients. Together, our findings demonstrate a critical role for TRPC1 in HSV-1 infection and suggest the channel as a potential target for anti-HSV therapy.
Asunto(s)
Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Canales Catiónicos TRPC/metabolismo , Internalización del Virus , Animales , Calcio/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Activación del Canal Iónico , Ratones , Modelos Biológicos , Mutación , Unión Proteica , Canales Catiónicos TRPC/genética , Células VeroRESUMEN
Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Compuestos de Boro/química , Compuestos de Boro/farmacología , Calcio/química , Retículo Endoplásmico/química , Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/química , Molécula de Interacción Estromal 2/genéticaRESUMEN
Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ entry pathway that is activated in response to depletion of ER-Ca2+ stores and critically controls the regulation of physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), which are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. While TRPC1 was associated with SOCE and regulation of function in several cell types, none of the TRPC members displayed I CRAC, the store-operated current identified in lymphocytes and mast cells. Intensive search finally led to the identification of Orai1 and STIM1 as the primary components of the CRAC channel. Orai1 was established as the pore-forming channel protein and STIM1 as the ER-Ca2+ sensor protein involved in activation of Orai1. STIM1 also activates TRPC1 via a distinct domain in its C-terminus. However, TRPC1 function depends on Orai1-mediated Ca2+ entry, which triggers recruitment of TRPC1 into the plasma membrane where it is activated by STIM1. TRPC1 and Orai1 form distinct store-operated Ca2+ channels that regulate specific cellular functions. It is now clearly established that regulation of TRPC1 trafficking can change plasma membrane levels of the channel, the phenotype of the store-operated Ca2+ current, as well as pattern of SOCE-mediated [Ca2+]i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca2+]i signal generated by Orai1. This review will highlight current concepts of the activation and regulation of TRPC1 channels and its impact on cell function.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Microdominios de Membrana/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Proteína ORAI1/metabolismoRESUMEN
Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.
Asunto(s)
Canales de Calcio/metabolismo , Caspasa 3/metabolismo , Radioterapia/efectos adversos , Glándulas Salivales/patología , Glándulas Salivales/efectos de la radiación , Molécula de Interacción Estromal 1/metabolismo , Células Acinares/metabolismo , Células Acinares/patología , Células Acinares/efectos de la radiación , Animales , Calcio/metabolismo , Canales de Calcio/genética , Caspasa 3/genética , Células Cultivadas , Humanos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/efectos de la radiación , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Glándulas Salivales/metabolismo , Molécula de Interacción Estromal 1/genética , Canales Catiónicos TRPM/metabolismo , Rayos XRESUMEN
Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ entry pathway that is activated in response to depletion of ER-Ca2+ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled ICRAC, the highly Ca2+-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca2+ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust ICRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca2+ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca2+ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca2+]i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca2+]i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Humanos , Transporte IónicoRESUMEN
Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteína ORAI1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Membrana Celular/genética , Retículo Endoplásmico/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo , Canales Catiónicos TRPC/genéticaAsunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Membrana Celular/metabolismo , Fenómenos Fisiológicos Celulares/fisiología , Retículo Endoplásmico/metabolismo , Humanos , MicroARNs/genética , Mitocondrias/metabolismoRESUMEN
Store-operated calcium entry (SOCE) is a ubiquitous Ca(2+) entry pathway that is activated in response to depletion of Ca(2+) stores within the endoplasmic reticulum (ER) and contributes to the control of various physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), that are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. TRPC channels show a miscellany of tissue expression, physiological functions and channel properties. However, none of the TRPC members display currents that resemble I CRAC. Intensive search for the CRAC channel component led to identification of Orai1 and STIM1, now established as being the primary constituents of the CRAC channel. There is now considerable evidence that STIM1 activates both Orai1 and TRPC1 via distinct domains in its C-terminus. Intriguingly, TRPC1 function is not only dependent on STIM1 but also requires Orai1. The critical functional interaction between TRPC1 and Orai1, which determines the activation of TRPC1, has also been identified. In this review, we will discuss current concepts regarding the role of TRPC channels in SOCE, the physiological functions regulated by TRPC-mediated SOCE, and the complex mechanisms underlying the regulation of TRPCs, including the functional interactions with Orai1 and STIM1.
Asunto(s)
Calcio/metabolismo , Canales Catiónicos TRPC/fisiología , Moléculas de Adhesión Celular/metabolismo , Humanos , Transporte Iónico , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , Canales Catiónicos TRPC/metabolismoRESUMEN
The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Células Epiteliales/metabolismo , Factores de Transcripción NFATC/metabolismo , Glándulas Salivales/metabolismo , Regulación hacia Arriba/fisiología , Acuaporina 5/biosíntesis , Acuaporina 5/genética , Canales de Calcio/biosíntesis , Células Cultivadas , Células Epiteliales/citología , Humanos , Factores de Transcripción NFATC/genética , Glándulas Salivales/citologíaRESUMEN
Studies over the past four decades have established that Ca(2+) is a critical factor in control of salivary gland function and have led to identification of the critical components of this process. The major ion transport mechanisms and ion channels that are involved in fluid secretion have also been established. The key event in activation of fluid secretion is an increase in [Ca(2+) ]i triggered by inositol 1,4,5-trisphosphate (IP3 )-induced release of Ca(2+) from ER via the IP3 receptor (IP3 R). IP3 Rs determine the site of initiation and the pattern of the [Ca(2+) ]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+) ]i and fluid secretion and is mediated by the store-operated Ca(2+) entry (SOCE) mechanism. Orai1, TRPC1, TRPC3 and STIM1 have been identified as critical components of SOCE in these cells. Cells finely tune the generation and amplification of [Ca(2+) ]i signals for regulation of cell function. An important emerging area is the concept that unregulated [Ca(2+) ]i signals in cells can directly cause cell damage, dysfunction and disease. Alternatively, aberrant [Ca(2+) ]i signals can also amplify and increase the rates of cell damage. Such defects in Ca(2+) signalling have been described in salivary glands in conjunction with radiation-induced loss of salivary gland function as well as in the salivary defects associated with the autoimmune exocrinopathy Sjögren's syndrome. Such defects have been associated with altered function or expression of key Ca(2+) signalling components, such as STIM proteins and TRP channels. These studies offer new avenues for examining the mechanisms underlying the disease and development of novel clinical targets and therapeutic strategies.
Asunto(s)
Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Glándulas Salivales/fisiología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/fisiopatología , Animales , Humanos , Glándulas Salivales/metabolismoRESUMEN
The autoimmune exocrinopathy, Sjögren's syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca(2+) release, critically required for Ca(2+) entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca(2+)]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca(2+) signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients.
Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Glándulas Salivales/metabolismo , Síndrome de Sjögren/patología , Células Acinares/citología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Estudios de Casos y Controles , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucinas/deficiencia , Interleucinas/genética , Linfotoxina-alfa/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Glándulas Salivales/patología , Síndrome de Sjögren/metabolismo , Proteínas de Transporte VesicularRESUMEN
Stromal interaction molecule 1 (STIM1) senses depletion of ER-Ca2+ store and clusters in ER-PM junctions where it associates with and gates Ca2+ influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2+ entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER-PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1-STIM1 clustering within ER-PM junctions following ER-Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells.
Asunto(s)
Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canales Catiónicos TRPC/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Membrana Celular/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Proteínas de Unión al GTP rab4/genética , Proteínas de Unión al GTP rab4/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.
Asunto(s)
Lisosomas/metabolismo , Proteolisis , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antifúngicos/farmacología , Línea Celular Tumoral , Humanos , Lisosomas/genética , Macrólidos/farmacología , Inhibidores de Proteasoma/farmacologíaRESUMEN
Store-operated calcium entry (SOCE) is activated within the endoplasmic reticulum (ER)-plasma membrane (PM) junctions in the plasma membrane and involves assembly of the channels, Orai1 and TRPC1, with the regulatory protein, STIM1, in response to depletion of Ca(2+) in the ER. The dynamic assembly of the channel complexes as well as regulation of channel activity and cell function is dependent on critical components that are either already localized in and/or recruited to the junctions following cell stimulation. These include proteins and lipids in the plasma membrane, ER, and cytosol. Together, these coordinated interactions lead to assembly and activation of Orai1/STIM1 and TRPC1/STIM1 channels. Recent studies demonstrate that Ca(2+) signals generated by Orai1 are detected locally within the ER-PM junctions by various signaling proteins that trigger and regulate other Ca(2+)-dependent functions. One such function is the plasma membrane recruitment of TRPC1 channels where it is activated by STIM1. Activation of TRPC1 within the ER-PM junctions leads to generation of distinct [Ca(2+)]i signals and regulation of cellular functions different from those regulated by Orai1. Thus, it has been suggested that Orai1 and TRPC1 channels are compartmentalized in distinct microdomains. Such compartmentalization involves organization of plasma membrane lipids as well as structural and scaffolding proteins, the nature of which is still poorly understood. Importantly, there appears to be dynamic reorganization of the microdomains within existing ER-PM junctions that allow segregated proteins to interact. Together, these highly specific and coordinated remodeling of protein complexes and membrane domains results in generation of spatially- and temporally-controlled Ca(2+) signals that are critical for numerous downstream cellular functions.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Microdominios de Membrana/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Membrana Celular/metabolismo , HumanosRESUMEN
A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists.