RESUMO
LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Receptores de Ácidos Lisofosfatídicos , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Endossomos/metabolismo , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/metabolismoRESUMO
Introduction: Cancer Stem Cells (CSC) are responsible for maintaining tumor growth, chemoresistance, and metastasis. Therefore, understanding their characteristics is critical to progress in cancer therapy. While the contribution of the canonical Wnt/b-catenin signaling in both normal and CSCs had been well established, the function of non-canonical Wnt signaling cascades in stem cells is unclear. Recently, we reported that Wnt ligands trigger complex signaling in which the canonical and non-canonical responses can be simultaneously activated by one ligand in colon cancer cells, suggesting, therefore, that noncanonical Wnt pathways may also be important in CSCs. Methods: The present work aimed to know the role of the Wnt/Ca2+ pathway in colon CSCs. We used tumorspheres as a model of CSCs enrichment of CRC cell lines with different Wnt/b-catenin contexts. Results: Using Wnt3a and Wnt5a as prototype ligands to activate the canonical or the non-canonical pathways, respectively, we found that both Wnt3a and Wnt5a promote sphere-formation capacity and proliferation without stimulating b-catenin-dependent transcription. Upregulation of sphere formation by Wnt5a or Wnt3a requires the downstream activation of Phospholipase C and transcriptional factor NFAT. Moreover, the single specific inhibition of PLC or NFAT, using U73122 and 11R-VIVIT, respectively, leads to impaired sphere formation. Discussion: Our results indicate that both types of ligands activate the Wnt/Ca2+ signaling axis to induce/maintain the self-renewal efficiency of CSCs, demonstrating to be essential for the functions of CSC in colon cancer.
RESUMO
Free fatty acid receptor 1 phosphorylation sites were studied using mutants, including a) a mutant with T215V in the third intracellular loop (3IL), b) another with changes in the carboxyl terminus (C-term): T287V, T293V, S298A, and c) a mutant with all of these changes (3IL/C-term). Agonist-induced increases in intracellular calcium were similar between cells expressing wild-type or mutant receptors. In contrast, agonist-induced FFA1 receptor phosphorylation was reduced in mutants compared to wild type. Phorbol ester-induced FFA1 receptor phosphorylation was rapid and robust in cells expressing the wild-type receptor and essentially abolished in the mutants. Agonist-induced ERK 1/2 phosphorylation and receptor internalization were decreased in cells expressing the mutant receptors compared to those expressing the wild-type receptor. Our data suggest that the identified sites might participate in receptor phosphorylation, signaling, and internalization.
Assuntos
Ácidos Graxos não Esterificados , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Mutação/genética , Fosforilação , Transdução de SinaisRESUMO
In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Ésteres de Forbol/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Endossomos/efeitos dos fármacos , Humanos , Proteínas Luminescentes , Metoxamina/farmacologia , Norepinefrina/farmacologia , Oximetazolina/farmacologia , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab5 de Ligação ao GTP/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Proteína Vermelha FluorescenteRESUMO
Sphingosine 1-phosphate (S1P) and FTY720-phosphate (FTYp) increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate (PMA) blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action of FTYp being more intense. S1P1 receptor-Rab protein (GFP-tagged) interaction was studied using FRET. The three agents were able to induce S1P1 receptor-Rab5 interaction, although with different time courses. S1P1 receptor-Rab9 interaction was mainly increased by the phorbol ester, whereas S1P1 receptor-Rab7 interaction was only increased by FTYp and after a 30-min incubation. These actions were not observed using dominant negative (GDP-bound) Rab protein mutants. The data suggested that the three agents induce interaction with early endosomes, but that the natural agonist induced rapid receptor recycling, whereas activation of protein kinase C favored interaction with late endosome and slow recycling and FTYp triggered receptor interaction with vesicles associated with proteasomal/lysosomal degradation. The ability of bisindolylmaleimide I and paroxetine to block some of these actions suggested the activation of protein kinase C was associated mainly with the action of PMA, whereas G protein-coupled receptor kinase (GRK) 2 (GRK2) was involved in the action of the three agents.