RESUMEN
Chemokine receptors play a central role in the maintenance of immune homeostasis and development of inflammation by directing leukocyte migration to tissues. GPR15 is a G protein-coupled receptor (GPCR) that was initially known as a co-receptor for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), with structural similarity to other members of the chemoattractant receptor family. Since the discovery of its novel function as a colon-homing receptor of T cells in mice a decade ago, GPR15 has been rapidly gaining attention for its involvement in a variety of inflammatory and immune disorders. The recent identification of its natural ligand C10orf99, a chemokine-like polypeptide strongly expressed in gastrointestinal tissues, has established that GPR15-C10orf99 is a novel signaling axis that controls intestinal homeostasis and inflammation through the migration of immune cells. In addition, it has been demonstrated that C10orf99-independent functions of GPR15 and GPR15-independent activities of C10orf99 also play significant roles in the pathophysiology. Therefore, GPR15 and its ligands are potential therapeutic targets. To provide a basis for the future development of GPR15- or GPR15 ligand-targeted therapeutics, we have summarized the latest advances in the role of GPR15 and its ligands in human diseases as well as the molecular mechanisms that regulate GPR15 expression and functions.
Asunto(s)
Receptores de Quimiocina , Receptores de Formil Péptido , Humanos , Ratones , Animales , Ligandos , Transducción de Señal , Inflamación , Receptores de Péptidos , Receptores Acoplados a Proteínas G/genéticaRESUMEN
We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.
Asunto(s)
Autofagia , Proteínas de Unión al GTP rab1 , Humanos , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Autofagosomas/metabolismoRESUMEN
GPR15 is a G-protein-coupled receptor (GPCR) that directs lymphocyte homing to the colon and skin. Recent studies have identified a chemokine-like protein GPR15L (also known as C10orf99) as a functional ligand of GPR15. In this study, we examined the structural elements that regulate the GPR15-GPR15L interaction with primary focus on post-translational modifications (PTMs) of receptor N-terminus and on the C-terminus of the ligand. Our findings reveal that the GPR15 receptor is sulfated on the N-terminal tyrosine residue(s) and disruption of tyrosine sulfation inhibits binding of GPR15L. In contrast, the disruption of O-glycosylation on the N-terminal threonine or serine residues, or the removal of α2,3-linked sialic acids from O-glycans, enhances the GPR15L binding. Thus, GPR15 represents a unique chemoattractant receptor in which different N-terminal PTMs regulate its ligand binding in a contrasting manner. We further demonstrate that, unlike canonical chemokines, GPR15L activity critically requires its extreme C-terminal residue and that its hydrophobicity may be a key attribute that facilitates an optimal interaction with the receptor. Our results reveal novel insights into chemoattractant receptor-ligand interaction and provide a valid footing for potential intervention targeting the GPR15-GPR15L axis.
Asunto(s)
Receptores de Formil Péptido , Tirosina , Glicosilación , Unión Proteica , Piel/metabolismo , Tirosina/metabolismoRESUMEN
Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
Asunto(s)
Tolerancia a Medicamentos , Proteínas de Unión al GTP/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/química , Transducción de Señal , Animales , Bencilaminas , Células CHO , Quimiocina CXCL12/metabolismo , Quimiotaxis , Cricetinae , Cricetulus , Ciclamas , Endocitosis , Fibroblastos/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Ligandos , Ratones , Fosforilación , Dominios Proteicos , Células THP-1 , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismoRESUMEN
Most newly synthesized proteins destined for the secretory pathway contain a signal peptide (SP) that triggers cotranslational translocation into the endoplasmic reticulum (ER). However, how small polypeptides undergo ER translocation is not fully understood. In this issue of JBC, Guo et al. describe a mechanism for posttranslational translocation of small secretory proteins featuring a positive charge within the SP N-terminal region. Defects in this element disrupt proper secretion and explain the effects of genetic mutations associated with one type of diabetes.
Asunto(s)
Péptidos/metabolismo , Proinsulina/química , Señales de Clasificación de Proteína , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Péptidos/química , Péptidos/genética , Proinsulina/genética , Proinsulina/metabolismo , Biosíntesis de Proteínas , Transporte de Proteínas , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
GPR15 is an orphan G protein-coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on ß-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the ß-arrestin- and GPCR kinase-dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.
Asunto(s)
Endocitosis/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Clatrina/metabolismo , Células HEK293 , Humanos , Ligandos , Mutación , Fosforilación , Unión Proteica , Dominios Proteicos , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
The activity of potassium (K(+)) channels critically depends on their density on the cell surface membrane, which is regulated by dynamic protein-protein interactions that often involve distinct trafficking signals on the cargo proteins. In this paper we explored the possibility of utilizing the Saccharomyces cerevisiae strain B31 for identification of the signal motifs that regulate surface expression of membrane proteins and for studying structure-function relationships of K(+) channels. B31 cells lack the K(+) efflux system and were reported to show overloaded K(+)-mediated growth inhibition in high K(+) media upon heterologous expression of a mammalian inwardly rectifying K(+) channel (Kir2.1). We show that while the expression of wild-type Kir2.1 channel inhibits the growth of B31 cells in high K(+) media, the human disease-causing mutations of Kir2.1 that abolish K(+) conduction (V302M) or surface trafficking (Δ314/315) fully restores the growth. The expression of two-pore-domain K(+) channel KCNK3 or KCNK9 also inhibited the growth of B31 in high K(+) media while C-terminal mutations that reduce their 14-3-3 protein-dependent cell surface trafficking restored the growth of B31. Finally, the expression of Kir2.1 channels that were C-terminally fused with known sequence motifs including ER retention/retrieval signals and an endocytosis signal allowed the growth of B31 in high K(+) media. These results demonstrate the potential of B31 yeast strain as a unique biological tool to screen the random peptide libraries for novel sequence signals that down-regulate surface expression of membrane proteins, as well as to systematically identify the structural determinants for cell surface trafficking and/or ion conductance of K(+) channels.
RESUMEN
Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg(310)-Arg(311)) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg(310)-Arg(311) in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs.
Asunto(s)
VIH/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Citometría de Flujo/métodos , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I(A)) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I(A) increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I(A) in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I(A) by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I(A) against ischemia.
Asunto(s)
Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Potasio/metabolismo , Regulación hacia Arriba , Animales , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Células Cultivadas , Femenino , Células HEK293 , Humanos , Canal de Potasio Kv1.4/genética , Canal de Potasio Kv1.4/metabolismo , Masculino , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/patología , Canales de Potasio con Entrada de Voltaje/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismoRESUMEN
Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores del VIH/metabolismo , Receptores de Péptidos/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutagénesis/fisiología , Fosforilación/fisiología , Estabilidad Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/química , Receptores de Péptidos/genética , Transducción de Señal/fisiologíaRESUMEN
The regulation of protein expression on the cell surface membrane is an important component of the cellular response to extracellular signalling. The translation of extracellular signalling into specific protein localization often involves the post-translational modification of cargo proteins. Using a genetic screen of random peptides, we have previously identified a group of C-terminal sequences, represented by RGRSWTY-COOH (termed'SWTY'), which are capable of overriding an endoplasmic reticulum localization signal and directing membrane proteins to the cell surface via specific binding to 14-3-3 proteins. The identity of the kinase signalling pathways that drive phosphorylation and 14-3-3 binding of the SWTY sequence is not known. In this study, we report that the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway by the over-expression of active kinases, stimulation with fetal bovine serum or growth factors can: (a) phosphorylate the SWTY sequence; (b) recruit 14-3-3 proteins to SWTY; and (c) promote surface expression of the chimeric potassium channel fused with the SWTY sequence. The expression of the dominant negative Akt inhibited the enhancement of surface expression by fetal bovine serum. In addition, the activation of PI3K significantly enhanced the 14-3-3 association and cell surface expression of GPR15, a G protein-coupled receptor which carries an endogenous SWTY-like, C-terminal, 14-3-3 binding sequence and is known to serve as a HIV co-receptor. Given the wealth and specificity of both kinase activity and 14-3-3 binding sequences, our results suggest that the C-terminal SWTYlike motif may serve as a sensor that can selectively induce the cell surface expression of membrane proteins in response to different extracellular signals.
Asunto(s)
Proteínas 14-3-3/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Bovinos , Línea Celular , Membrana Celular/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Fosforilación , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
The pore-forming subunit of the large-conductance Ca(2+)-dependent K(+) (Slo1) channel is encoded by one gene. However, the functional properties of Slo1 channels are diverse in part because of their numerous regulatory mechanisms including posttranslational modification and alternative splicing. In particular, multiple splice variants of the pore-forming subunit have been reported but their significance is only beginning to be elucidated. Here we examined the cell biological properties of the three common C-terminal isoforms that differ in the last 8 (Slo1_ERL and Slo1_VYR) or 61 residues (Slo1_DEC). We found that Slo1_DEC, the longest isoform, shows dramatically reduced surface expression compared to that of Slo1_ERL or Slo1_VYR. Immunocytochemistry revealed that a large fraction of Slo1_DEC remains localized in endoplasmic reticulum (ER). Using a GST fusion protein containing the Slo1_DEC-specific sequence, affinity purification was carried out to isolate interacting proteins. The identified proteins include protein phosphatase 2A (PP2A-A), actin, and tubulin. The PP2A-A interaction is specific to Slo1_DEC and causes a significant reduction of phosphorylation in Slo1_DEC but not Slo1_ERL or Slo1_VYR. The results together support the notion that Slo1_DEC nucleates isoform-specific protein complexes and possesses a cis element(s) for regulating trafficking of the Slo1 channels.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Células COS , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Activación del Canal Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteómica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.
Asunto(s)
Proteínas 14-3-3/inmunología , Proteínas 14-3-3/metabolismo , Anticuerpos Fosfo-Específicos/inmunología , Técnicas Biosensibles/métodos , Estructura Molecular , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Sensibilidad y EspecificidadRESUMEN
The density and composition of cell surface proteins are major determinants for cellular functions. Regulation of cell surface molecules occurs at several levels, including the efficiency of surface transport, and is therefore of great interest. As the major phosphoprotein-binding modules, 14-3-3 proteins are known for their crucial roles in a wide range of cellular activities, including the subcellular localization of target proteins. Accumulating evidence suggests a role for 14-3-3 in surface transport of membrane proteins, in which 14-3-3 binding reduces endoplasmic reticulum (ER) localization, thereby promoting surface expression of membrane proteins. Here, we focus on recent evidence of 14-3-3-mediated surface transport and discuss the possible molecular mechanisms.
Asunto(s)
Proteínas 14-3-3/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Animales , Sitios de Unión , Membrana Celular/metabolismo , Unión Proteica , Señales de Clasificación de ProteínaRESUMEN
Amino and carboxyl termini are unique positions in a polypeptide. They tend to be exposed in folded three dimensional structures. Diversity and functional significance of C-terminal sequences have been appreciated from studies of PDZ and PEX domains. Signaling 14-3-3 protein signaling by recognizing phosphorylated peptides plays a critical role in a variety of biological processes, including oncogenesis. The preferential binding of 14-3-3 to phosphorylated C-terminal sequences, mode III, provides a means of regulated binding and considerably expands the substrate repertoire of 14-3-3 interaction partners.
Asunto(s)
Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Secuencia de Consenso , Humanos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
A high percentage of drugs and drug candidates has been found to cause cardiotoxicity by reducing potassium conductance, more commonly known as QT prolongation. However, some compounds do not show direct block of ionic flow, suggesting that other mechanisms may also lead to reduction of potassium currents. Using the functional recovery after chemobleaching (FRAC) assay, we have examined a collection of drugs and drug-like compounds for potential perturbation of cardiac potassium channel trafficking. Here we report that a significant number of inhibitory compounds displayed effects on channel expression on the cell surface. Further investigation of celastrol (3-hydroxy-24-nor-2-oxo-1 (10),3,5,7-friedelatetraen-29-oic acid), a cell-permeable dienonephenolic triterpene compound, revealed its potent inhibitory activity on both Kir2.1 and hERG potassium channels, causal to QT prolongation. In addition to acute block of ion conduction, celastrol also alters the rate of ion channel transport and causes a reduction of channel density on the cell surface. In contrast, celastrol has no effects on trafficking of either CD4 or CD8 membrane proteins. Furthermore, the potency for reducing surface expression is approximately 5-10-fold more effective than that for either direct acute inhibition or reported cytoprotectivity via activation of the heat shock transcription factor 1. Because the reduction of potassium channel activity is a common form of druginduced cardiotoxicity, the potent inhibition of cell surface expression by celastrol underscores a need to evaluate drug candidates for their chronic effects on biogenesis of potassium channels. Our results suggest that chronic exposure to certain drugs may be an important aspect of acquired QT prolongation.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Triterpenos/metabolismo , Acetatos , Animales , Antipsicóticos/química , Antipsicóticos/metabolismo , Línea Celular , Clorpromazina/química , Clorpromazina/metabolismo , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Estructura Molecular , Técnicas de Placa-Clamp , Triterpenos Pentacíclicos , Canales de Potasio de Rectificación Interna/genética , Congéneres de la Progesterona , Transporte de Proteínas/fisiología , Reproducibilidad de los Resultados , Rubidio/metabolismo , Canales de Potasio Shab/metabolismo , Triterpenos/químicaRESUMEN
Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.
Asunto(s)
Proteínas 14-3-3/metabolismo , Sondas Moleculares , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Unión Proteica , Sensibilidad y Especificidad , SolucionesRESUMEN
Dendritic cells (DC) are far more potent to activate T cells than other antigen presenting cells (e.g., macrophages) and distributed to many organs where DC develop to functionally and phenotypically distinctive subsets. To isolate DC-differentially expressed genes, we used a subtractive cDNA cloning (XS52 DC minus J774 macrophages), resulting in the identification of d2 isoform of vacuolar (V) H+-ATPase subunit d. Unlike the ubiquitously expressed isoform (d1), d2 mRNA manifested expression restricted to particular subsets of DC (e.g., skin- and bone marrow-derived DC) among leukocytes and encoded two transcripts (1.6 and 3.0 kb) that differed in the length of the 3'-untranslated region. The d2 protein displayed association with membranes and the localization in lysosomes and antigen-containing endosomes. Interestingly, XS52 DC expressed seven-fold higher V-ATPase proton-pump activity than J774 macrophages and distinguished from the macrophage by high levels of isoforms a1 and a2 expression among V-ATPase subunits. These results indicated that d2 is a new marker for DC and it may, co-operatively with subunit a isoforms, regulate V-ATPase activity.
Asunto(s)
Células Dendríticas/enzimología , Células Dendríticas/inmunología , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Presentación de Antígeno , Secuencia de Bases , Línea Celular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Células Dendríticas/clasificación , Endosomas/enzimología , Endosomas/inmunología , Expresión Génica , Técnicas In Vitro , Leucocitos/clasificación , Leucocitos/enzimología , Leucocitos/inmunología , Activación de Linfocitos , Macrófagos/enzimología , Macrófagos/inmunología , Membranas/enzimología , Ratones , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/inmunologíaRESUMEN
Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.
Asunto(s)
Membrana Celular/fisiología , Canales de Potasio de Rectificación Interna/metabolismo , Transducción de Señal/genética , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos/fisiología , Línea Celular , Membrana Celular/genética , Retículo Endoplásmico/metabolismo , Humanos , Canales de Potasio de Rectificación Interna/genética , Transporte de Proteínas/fisiologíaRESUMEN
Diverse functions of 14-3-3 proteins are directly coupled to their ability to interact with targeted peptide substrates. RSX(pS/pT)XP and RXPhiX(pS/pT)XP are two canonical consensus binding motifs for 14-3-3 proteins representing the two common binding modes, modes I and II, between 14-3-3 and internal peptides. Using a genetic selection, we have screened a random peptide library and identified a group of C-terminal motifs, termed SWTY, capable of overriding an endoplasmic reticulum localization signal and redirecting membrane proteins to cell surface. Here we report that the C-terminal SWTY motif, although different from mode I and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant (K(D)) of 0.17 microM, comparable with that of internal canonical binding peptides. We show that all residues but proline in -SWTX-COOH are compatible for the interaction and surface expression. Because SWTY-like sequences have been found in native proteins, these results support a broad significance of 14-3-3 interaction with protein C termini. The C-terminal binding consensus, mode III, represents an expansion of the repertoire of 14-3-3-targeted sequences.