Exogenous myristoylated-G(i2)alpha subunits of GTP-binding proteins are mitogens following their internalization by astrocytes in culture.
Heterotrimeric GTP-binding proteins (G proteins) are involved in the coupling of a variety of cell surface receptors to different intracellular signalling pathways, some of which take part in the regulation of growth by affecting cell proliferation and/or differentiation. In cultured astrocytes, many receptors of neuropeptides and hormones are coupled to the heterotrimeric G(i) proteins which regulate the mitogen-activated protein kinase (MAPK/ERK) cascade through both the Galpha and Gbetagamma subunits. We have previously reported that functionally active recombinant myr-G(i2)alpha subunits added to such cultures are internalised and distributed within the plasma membrane and cytosol as well as in the nuclei of dividing astrocytes. Here we show that astrocytes proliferate dose-dependently in response to exogenous myr-G(i2)alpha subunits. Concentrations of 100 pM-30 nM myr-G(i2)alpha caused more than 2.5-fold increase of [3H]thymidine incorporation over basal levels. Other classes of myr-Galpha subunits, such as G(i3)alpha or G(o)alpha, induced a much lower proliferative effect. The addition of G(i1)alpha subunits to the cultures produced no change, indicating the selectivity of this effect. Even though myr-G(i2)alpha subunits are internalised by the cells regardless of their guanine nucleotide-bound state, much less [3H]thymidine incorporation was observed in the presence of GDPbetaS-myr-G(i2)alpha or GTPgammaS-myr-G(i2)alpha. Further, the fluorescent labelling was dissimilarly distributed, the signal being concentrated in the nucleus and perinuclear regions of the astrocytes. Selective disassembly of caveolae impaired both myr-G(i2)alpha internalisation and DNA induction. Together, these data reveal a proliferative effect of myr-G(i2)alpha subunits in astrocytes, and provide evidence for the incorporation of exogenous myr-G(i2)alpha subunits into the mitogen cascade activated by neurotransmitters or growth factors. The fact that Galpha proteins can enter cells is particularly interesting because options for delivering functional proteins into cells are limited. Thus, these proteins may have clinical applications for compensating deficits in the transduction mechanisms associated with several neurological diseases, or as a non-invasive membrane traversing carriers.
Sánchez-Blázquez
Pilar
P
Neuropharmacology, Instituto Neurobiología Santiago Ramón y Cajal, CSIC, Avenida Doctor Arce 37, 28002 Madrid, Spain. psb@cajal.csic.es
De Antonio
Isabel
I
Montero
Carlos
C
Garzón
Javier
J
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D005229:Fatty Acids, Monounsaturated / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000493:pharmacokinetics*; D008934:Mitogens / Q000493:pharmacokinetics*; D011518:Proto-Oncogene Proteins / Q000493:pharmacokinetics*; D051381:Rats; D017208:Rats, Wistar
D001798:Blood Proteins; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008934:Mitogens; D011518:Proto-Oncogene Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; C054211:9-tetradecenoic acid