Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.

• Klíčová slova
• Adrenergic Receptor, Cell Signaling, Heterotrimeric G Proteins, Membrane Proteins, Microscopic Imaging, Plasma Membrane, Potassium Channels, Two-photon Polarization Microscopy,
• MeSH
• aktivace enzymů fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• protein GAP-43 genetika   metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go genetika   metabolismus   MeSH
• proteiny vázající GTP - beta-podjednotky genetika   metabolismus   MeSH
• proteiny vázající GTP - gama-podjednotky genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein GAP-43 MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
• proteiny vázající GTP - beta-podjednotky MeSH
• proteiny vázající GTP - gama-podjednotky MeSH

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.

• MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• konformace proteinů MeSH
• membránové proteiny metabolismus   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• polarizační mikroskopie metody   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH