RESUMEN
LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid ß-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute ß-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.
Asunto(s)
Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , beta-Arrestinas/metabolismo , Sitios de Unión , Señalización del Calcio , Células HEK293 , Lisofosfolípidos/metabolismo , Fosforilación , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with ß-arrestin 2 and LPA3 internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA3 receptor function.
Asunto(s)
Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , Fosforilación , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Células HEK293 , Lisofosfolípidos/metabolismo , Calcio/metabolismo , Endocitosis , MutaciónRESUMEN
The function of the α1B-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α1B-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.
Asunto(s)
Calcio , Norepinefrina , Fosforilación , Calcio/metabolismo , Norepinefrina/farmacología , Ésteres del Forbol , Receptores Adrenérgicos/metabolismoRESUMEN
MicroRNAs are heterochronic molecules important during brain development, which could be altered by gestational diabetes mellitus (GDM). To explore these molecules in maternal serum, we performed an RT-qPCR analysis. Our results revealed the heterochronic character of some neural development-related microRNA in serum samples of pregnant women. In relation to the first trimester, higher levels of miR-183-5p, -200b-3p, and -125-5p in the second trimester, and higher levels of miR-137 in the third trimester, were found. Furthermore, an insult such as GDM led to higher levels of miR-183-5p, -200b-3p, -125-5p, and -1290 relative to the control in the first trimester, which might be related to changes in neurogenesis and cell proliferation. An in silico analysis suggested that increased microRNAs in the second trimester in the control contributed to cell proliferation and neuron differentiation and that the rise in miR-137 in the third trimester led to neuron maturation. In the diabetic, higher levels of the microRNAs in the first trimester suggested alterations in cell proliferation and neuron differentiation. In conclusion, we showed that fetal-related microRNAs can be detected in the serum of pregnant woman and exhibit temporary regulation during pregnancy and that microRNAs involved in cell proliferation and neuron differentiation are upregulated under GDM.
Asunto(s)
Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Diabetes Gestacional/sangre , MicroARNs/sangre , Adolescente , Adulto , Simulación por Computador , Femenino , Humanos , Embarazo , Primer Trimestre del Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Adulto JovenRESUMEN
Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 µM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.