Identification and isolation of common and tissue‐specific geranylgeranylated γ subunits of guanine‐nucleotide‐binding regulatory proteins in various tissues

Abstract

Hetcrotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their tl subunits, though a notable multiplicity of ;J subunits has also been demonstrated. To investigate whether each subtype of a subunit is associated with a particular y 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 y subunit. Of the brain G proteins, G,A, GOB, and Cil contain the same set of three y subunits, but Gi2 contains only two or these subunits. Lung GiI and Giz and spleen Giz and Gi3 had similar sets of two y subunits, one of which was distinct from the y subunits of brain G proteins. These observations indicate that each subtype of a subunit is associated with a variety of By subunits, and that the combinations differ among cells. For analyses of the structural diversity of the y subunits, py subunits were purified from the total G proteins of each tissue and subjcctcd to reverse-phase HPLC under denaturing conditions, where none of the p subunits were eluted from the column. Three distinct y subunits were isolated in this way from brain p1) subunits. In contrast, lung and spleen subunits contained at least five y subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several y 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 y subunit in each tissue was identical, and the sequences coincided exactly with that of 'y6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1 989) J . Riol. Chem. 264, IS 758 -15 7611. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant 7 subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'ys' from brain. In addition to abundant y subunits, other tissue-specific y subunits were also shown to be geranylgeranylatcd by gas-chromatographycoupled mass spectrometry analysis of Raney nickel-treated 1' subunits. These results suggest that most y subunits associated with many different subtypes of ci subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated y subunit.

G Protein γ Subunits with Altered Prenylation Sequences Are Properly Modified When Expressed in Sf9 Cells

“…Under the conditions of the separation, the ␥ subunit is eluted in a well defined peak, but we and others (33)(34)(35)(36) have been unable to detect the elution of the ␤ subunit. Presumably the acidic conditions of the separation result in not only the dissociation of the ␤␥ dimer but also in the irreversible adsorption of the denatured ␤ subunit to the hydrophobic stationary phase.…”

Selective Association of G Protein β4 with γ5 and γ12 Subunits in Bovine Tissues

“…4). However, the fractions immunoprecipitated from the brain with antibodies against ␥ 5 and ␥ 12 contained small amounts of ␥ 2 and ␥ 7 , probably due to the high concentrations of ␥ 2 and ␥ 7 , in contrast to the low concentrations of ␥ 5 and ␥ 12 , in the bovine brain (24,30). Analyses of the individual ␤ subunits revealed remarkable variations in the levels of ␤ 4 among the immunoprecipitates.…”

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

“…By contrast, 72,74,7~,7i' Yio,'Yii~and 712 are distributed in a variety of tissues (Gautam et al, 1990;Call et al, 1992;Morishita et al, 1992Morishita et al, , 1995Asano et al, 1995;Ray et al, 1995). Such tissuespecific distribution of some of the y subunits suggests that functional differences might exist among these subunits.…”

“…y~is localized only in the brain (Gautam et al, 1990;Cali et al, 1992;Morishita et al, 1992;Asano et al, 1995). By contrast, 72,74,7~,7i' Yio,'Yii~and 712 are distributed in a variety of tissues (Gautam et al, 1990;Call et al, 1992;Morishita et al, 1992Morishita et al, , 1995Asano et al, 1995;Ray et al, 1995).…”