Assays of nuclear localization of R7/Gbeta5 complexes.
Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein signaling proteins and the Gbeta5 isoform of the heterotrimeric G-protein beta subunit family are strongly expressed in the cell nucleus in neurons and brain, as well as in the cytoplasm and plasma membrane. Native and recombinant Gbeta5 and/or R7 expression have been studied in model systems like rat pheochromocytoma PC12 cells where their nuclear localization can be studied by fluorescence microscopy and/or subcellular fractionation. Nucleic acid counterstains chosen for compatibility with the fluorescent tags on secondary antibodies can facilitate the assay of R7/Gbeta5 nuclear localization by epifluorescence or confocal laser microscopy. Subcellular fractionation allows isolation of a purified nuclear fraction that can be probed for the presence of Gbeta5 and/or R7 subunits by immunoblots or immunoprecipitation and compared to other subcellular fractions. While the function of nuclear R7/Gbeta5 complexes is unknown, comparison with the properties of other RGS proteins that localize to the cell nucleus may suggest modes of action. Models are offered in which the reversible post-translational modification of R7/Gbeta5 complexes regulates their nuclear localization and signaling activity, whether the target of such signaling activity is in the nucleus, at the plasma membrane, or both.
Metabolic Diseases Branch/NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
William F
WF
Simonds
Geoffrey E
GE
Woodard
Jian-Hua
JH
Zhang
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D016716:PC12 Cells; D020033:Protein Isoforms / Q000378:metabolism; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D044387:GTP-Binding Protein beta Subunits; C490610:Gnb5 protein, rat; D046911:Macromolecular Substances; D020033:Protein Isoforms; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins