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

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Abstract
Heterotrimeric guanine‐nucleotide‐binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their α subunits, though a notable multiplicity of γ subunits has also been demonstrated. To investigate whether each subtype of α subunit is associated with a particular γ 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 tow kinds of γ subunit. Of the brain G proteins, GoA, GoB, and Gil contain the same set of three γ subunits, but Gi2 contains only two of these subunits. Lung Gil and Gi2 and spleen Gi2 and Gi3 had similar sets of two γ subunits, one of which was distinct from the γ subunits of brain G proteins. These observations indicate indicate that each subtype of α subunit is associated with a variety of βγ subunits, and that the combinations differ among cells. For analyses of the structural diversity of the γ subunits, βγ subunits were purified from the total G proteins of each tissue and subjected to reverse‐phase HPLC under denaturing conditions, where none of the β subunits were eluted from the column. Three distincy γ subunits were isolated in this way from brain βγ subunits. In contrast, lung and spleen βγ subunits contained at least five γ subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several γ 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 γ subunit in each tissue was identical, and the sequences coincided exactly with that of ‘γ6’ [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 γ subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C‐terminus, as was ‘γ6’ from brain. In addition to abundant γ subunits, other tissue‐specific γ subunits were also shown to be geranylgeranylated by gas‐chromatographycoupled mass spectrometry analysis of Raney nickel‐treated γ subunits. These results suggest that most γ subunits associated with many different subtypes of α subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated γ subunit.