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 Gi/Go 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.

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