Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.
Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.
Morishita
R
R
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Fukada
Y
Y
Kokame
K
K
Yoshizawa
T
T
Masuda
K
K
Niwa
M
M
Kato
K
K
Asano
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004224:Diterpenes / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008401:Gas Chromatography-Mass Spectrometry; D008168:Lung / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013154:Spleen / Q000378:metabolism
D004224:Diterpenes; D046911:Macromolecular Substances; D010446:Peptide Fragments; C017338:geranylgeraniol; D019204:GTP-Binding Proteins