Species- and tissue-dependent diversity of G-protein β subunit phosphorylation: evidence for a cofactor

We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein β subunit (Gβ) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111–18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least Gβ₁ and Gβ₂ subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly Gβ₁ or both subtypes at roughly equal concentrations. In contrast, significantly more Gβ₂ immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak Gβ-specific immunoreactive signals. Conversely, these membranes showed the highest levels of Gβ phosphorylation after incubation with [γ-³²P]GTP or ³⁵S-labelled guanosine 5´-[γ-thio]triphosphate. Interestingly, Gβ-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. Gβ phosphorylation was confirmed by immunoprecipitation with Gβ-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated Gβ-proteins differed in their apparent molecular size from unmodified Gβ-proteins. Moreover, phosphorylated Gβ-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish Gβ phosphorylation. In contrast, reconstituted purified G_i/G_o proteins showed no Gβ phosphorylation. From these experiments we conclude that: (i) Gβ phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated Gβ-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) Gβ phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.