RESUMEN
TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.
Asunto(s)
Fosfoserina/metabolismo , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Células HEK293 , Humanos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación/efectos de los fármacos , Fosfoserina/química , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Coloración y Etiquetado , Canal Catiónico TRPC6RESUMEN
TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).
Asunto(s)
Miocitos del Músculo Liso/metabolismo , Proteína Quinasa C-delta/metabolismo , Canales Catiónicos TRPC/metabolismo , Sustitución de Aminoácidos , Carbacol/farmacología , Carcinógenos/farmacología , Proliferación Celular/efectos de los fármacos , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Mióticos/farmacología , Mutación Missense , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Serina/genética , Serina/metabolismo , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Acetato de Tetradecanoilforbol/farmacología , Vasoconstrictores/farmacología , Vasopresinas/farmacologíaRESUMEN
TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface.
Asunto(s)
Canales Catiónicos TRPC/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Proteínas de Unión al GTP rab/genéticaRESUMEN
Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.
Asunto(s)
Señalización del Calcio , Caveolas/metabolismo , Caveolina 1/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor de Activación Plaquetaria/fisiología , Animales , Sitios de Unión/inmunología , Células CHO , Señalización del Calcio/inmunología , Caveolas/enzimología , Caveolas/inmunología , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Activación Enzimática/inmunología , Humanos , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
The recessive lyp allele, which harbors a defective gimap5 (GTPase of immunity-associated nucleotide binding protein 5) gene, causes spontaneous apoptosis of T lymphocytes in the biobreeding diabetes-prone strain of rats. Mechanisms underlying the pro-survival function of GIMAP5 remain unclear. In this study, we show that gimap5(lyp/lyp) T cells display diminished calcium flux in response to thapsigargin or signaling via the T cell antigen receptor. This defect is manifested in mature single positive thymocytes, where the survival defect first occurs. We also show that GIMAP5 deficiency does not affect the thapsigargin-induced calcium release from the intracellular stores but impairs subsequent calcium entry across the plasma membrane. Our findings suggest that GIMAP5 is an important regulator of calcium response in T lymphocytes and impaired calcium signaling might underlie spontaneous apoptosis of gimap5(lyp/lyp) T cells.
Asunto(s)
Señalización del Calcio/inmunología , Proteínas de Unión al GTP/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Animales Modificados Genéticamente , Señalización del Calcio/fisiología , Membrana Celular/fisiología , Proteínas de Unión al GTP/genética , Técnicas In Vitro , Ratas , Linfocitos T/efectos de los fármacos , Tapsigargina/farmacología , Timo/citologíaRESUMEN
TRPCs function as cation channels in non-excitable cells. The N-terminal tails of all TRPCs contain an ankyrin-like repeat domain, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid screening approach, we found that RNF24, a new membrane RING-H2 protein, interacted with the ankyrin-like repeat domain of TRPC6. GST pull-down and co-immunoprecipitation assays showed that RNF24 interacted with all TRPCs. Cell surface-labelling assays showed that the expression of TRPC6 at the surface of HEK 293T cells was greatly reduced when it was transiently co-transfected with RNF24. Confocal microscopy showed that TRPC3 and TRPC6 co-localized with RNF24 in a perinuclear compartment and that RNF24 co-localized with mannosidase II, a marker of the Golgi cisternae. Using a pulse-chase approach, we showed that RNF24 did not alter the maturation process of TRPC6. Moreover, in HEK 293T cells, RNF24 did not alter carbachol-induced Ca(2+) entry via endogenous channels or TRPC6. These results indicate that RNF24 interacts with TRPCs in the Golgi apparatus and affects TRPC intracellular trafficking without affecting their activity.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Repetición de Anquirina , Carbacol/farmacología , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Línea Celular , Membrana Celular/metabolismo , Aparato de Golgi/química , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Canales Catiónicos TRPC/química , Canal Catiónico TRPC6 , Ubiquitina-Proteína LigasasRESUMEN
Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.
Asunto(s)
Calcio/metabolismo , Canales de Potencial de Receptor Transitorio/química , Animales , Línea Celular , Citosol/metabolismo , Electrofisiología , Endopeptidasa K/química , Glutatión Transferasa/metabolismo , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Transfección , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
TRPC proteins are the mammalian homologues of the Drosophila transient receptor potential channel and are involved in calcium entry after agonist stimulation of non-excitable cells. Seven mammalian TRPCs have been cloned, and their mechanisms of activation and regulation are still the subject of intense research. TRPC proteins interact with the inositol 1,4,5-trisphosphate receptor, and the conformational coupling plays a critical role in the activation of calcium entry. Some evidence also supports an exocytotic mechanism as part of the activation of calcium entry. To investigate the possible involvement of exocytosis in TRPC6 activation, we evaluated the location of TRPC6 at the plasma membrane by biotinylation labeling of cell surface proteins and by indirect immunofluorescence marking of TRPC6 in stably transfected HEK 293 cells. We showed that when the muscarinic receptor was stimulated or the thapsigargin-induced intracellular calcium pool was depleted the level of TRPC6 at the plasma membrane increased. The carbachol concentration at which TRPC6 externalization occurred was lower than the concentration required to activate TRPC6. Externalization occurred within the first 30 s of stimulation, and TRPC6 remained at the plasma membrane as long as the stimulus was present. These results indicate that an exocytotic mechanism is involved in the activation of TRPC6.