RESUMO
Heterotrimeric G proteins of the G(i) class have been implicated in signaling pathways regulating growth and metabolism under physiological and pathophysiological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed Galpha(i) class genes, Galpha(i2) and Galpha(i3), demonstrate shared as well as gene-specific functions. The presence of a single active allele of Galpha(i3) is sufficient for embryonic development, whereas at least one allele of Galpha(i2) is required for extrauterine life. Mice lacking both Galpha(i2) and Galpha(i3) are massively growth-retarded and die in utero. We have used biochemical and cell biological methods together with in situ liver perfusion experiments to study Galpha(i) isoform-specific functions in Galpha(i2)- and Galpha(i3)-deficient mice. The subcellular localization of Galpha(i3) in isolated mouse hepatocytes depends on the cellular metabolic status. Galpha(i3) localizes to autophagosomes upon starvation-induced autophagy and distributes to the plasma membrane upon insulin stimulation. Analysis of autophagic proteolysis in perfused mouse livers showed that mice lacking Galpha(i3) are deficient in the inhibitory action of insulin. These data indicate that Galpha(i3) is crucial for the antiautophagic action of insulin and suggest an as-yet-unrecognized function for Galpha(i3) on autophagosomal membranes.
Assuntos
Autofagia/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Anticorpos/farmacologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/imunologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Masculino , Camundongos , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacosRESUMO
Cell migration plays a central role in many physiological and pathophysiological processes. On a cellular level it is based on a highly coordinated restructuring of the cytoskeleton, a continuous cycle of adhesion and de-adhesion as well as on the activity of ion channels and transporters. The cytoplasmic Ca2+ ([Ca2+]i) concentration is an important coordinator of these intracellular processes. Thus, [Ca2+]i must be tightly controlled in migrating cells. This is among other things achieved by the activity of Ca2+ permeable channels, the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) in the plasma membrane. Here, we wanted to determine the functional role of these transport proteins in cell migration. We therefore quantified the acute effect of inhibitors of these transport proteins (Gd3+, vanadate, KB-R7943) on migration, [Ca2+]i, and intracellular pH (pHi) of MDCK-F cells. Migration was monitored with computer-assisted time-lapse video microscopy. [Ca2+]i and pHi were measured with the fluorescent indicators fura-2 and BCECF. NCX expression in MDCK-F cells was verified with ion substitution experiments, and expression of PMCA was tested with RT-PCR. All blockers lead to a rapid impairment of cell migration. However, the most prominent effect is elicited by NCX-inhibition with KB-R7943. NCX-blockade leads to an almost complete inhibition of migration which is accompanied by a dose-dependent increase of [Ca2+]i and an intracellular alkalinisation. We show that inhibition of NCX and PMCA strongly affects lamellipodial dynamics of migrating MDCK-F cells. Taken together, our results show that PMCA and in particular NCX are of critical importance for cell migration.