Species- and tissue-dependent diversity of G-protein beta 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 beta subunit (G beta) 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 G beta 1 and G beta 2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly G beta 1 or both subtypes at roughly equal concentrations. In contrast, significantly more G beta 2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak, G beta-specific immunoreactive signals. Conversely, these membranes showed the highest levels of G beta phosphorylation after incubation with [gamma-32P]GTP or 35S-labelled guanosine 5'-[gamma-thio]triphosphate. Interestingly, G beta-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. G beta phosphorylation was confirmed by immunoprecipitation with G beta-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated G beta-proteins differed in their apparent molecular size from unmodified G beta-proteins. Moreover, phosphorylated G beta-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish G beta phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no G beta phosphorylation. From these experiments we conclude that: (i) G beta phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated G beta-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) G beta phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.
Nürnberg
B
B
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Harhammer
R
R
Exner
T
T
Schulze
R A
RA
Wieland
T
T
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D009928:Organ Specificity; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D013045:Species Specificity; D013462:Sulfur Radioisotopes; D013552:Swine
D046911:Macromolecular Substances; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins