Thiophosphorylation of the G protein beta subunit in human platelet membranes: evidence against a direct phosphate transfer reaction to G alpha subunits

M Hohenegger, T Mitterauer, T Voss, C Nanoff and M Freissmuth

Institute of Pharmacology, Vienna University, Austria.

A direct phosphate transfer reaction from the G protein beta subunits to either Gs alpha or Gi alpha has been proposed to account for the ability of thiophosphorylated transducin beta gamma-dimers to bidirectionally regulate adenylyl cyclase activity in human platelet membranes. We searched for experimental evidence for this reaction. Incubation of human platelet membranes with [35S]guanosine-5'-(3-O- thio)triphosphate ([35S]GTP gamma S) results in the predominant incorporation of [35S]thiophosphate into a 36-kDa protein, which comigrates with the G protein beta subunit and is immunoprecipitated by a beta subunit-specific antiserum. Thiophosphorylation of the beta subunit is specific for guanine nucleotides and abolished by the histidine-modifying agent diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of [35S]thiophosphorylated beta subunits is accelerated in the presence of GDP, but not ADP, UDP, or guanosine- 5'-(2-O-thio)diphosphate. Neither the thiophosphorylation nor the dephosphorylation is sensitive to receptor agonists (alpha 2- adrenergic, A2 adenosine, thrombin, or insulin), and purified G protein alpha subunits do not act as thiophosphate donors. An approach was designed to demonstrate direct thiophosphate transfer to protein-bound nucleotides; platelet membranes were sequentially exposed to NaIO4, NaCNBH3, and NaBH4, an oxidation-reduction step that covalently incorporates prebound nucleotides into proteins. Under these conditions, multiple radiolabeled proteins are visualized on subsequent addition of [35S]GTP gamma S. This reaction is specific because both oxidation and reduction are required and pretreatment of platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyrocarbonate blocks the subsequent labeling in oxidized and reduced membranes. The G protein beta subunit may participate in this thiophosphate transfer reaction. Most important, however, no labeled G protein alpha subunits (Gs alpha and Gi alpha) were recovered by immunoprecipitation from oxidized and reduced membranes subsequent to the addition of [35S]GTP gamma S. Thus, our results clearly rule out the existence of a postulated G protein activation by phosphate transfer reactions, which lead to the formation of GTP from GDP prebound to the alpha subunit.

Volume 49, Issue 1, pp. 73-80, 01/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				F. Cuello, R. A. Schulze, F. Heemeyer, H. E. Meyer, S. Lutz, K. H. Jakobs, F. Niroomand, and T. Wieland Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gbeta Subunits. COMPLEX FORMATION OF NDPK B WITH Gbeta gamma DIMERS AND PHOSPHORYLATION OF His-266 IN Gbeta J. Biol. Chem., February 21, 2003; 278(9): 7220 - 7226. [Abstract] [Full Text] [PDF]

				H.-J. Hippe, S. Lutz, F. Cuello, K. Knorr, A. Vogt, K. H. Jakobs, T. Wieland, and F. Niroomand Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gbeta Subunits. SPECIFIC ACTIVATION OF Gsalpha BY AN NDPK B{middle dot}Gbeta gamma COMPLEX IN H10 CELLS J. Biol. Chem., February 21, 2003; 278(9): 7227 - 7233. [Abstract] [Full Text] [PDF]

				M. T. Hartsough, D. K. Morrison, M. Salerno, D. Palmieri, T. Ouatas, M. Mair, J. Patrick, and P. S. Steeg Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway J. Biol. Chem., August 23, 2002; 277(35): 32389 - 32399. [Abstract] [Full Text] [PDF]

				M. Waldhoer, E. Bofill-Cardona, G. Milligan, M. Freissmuth, and C. Nanoff Differential Uncoupling of A1 Adenosine and D2 Dopamine Receptors by Suramin and Didemethylated Suramin (NF037) Mol. Pharmacol., May 1, 1998; 53(5): 808 - 818. [Abstract] [Full Text]

				E. Bofill-Cardona, O. Kudlacek, Q. Yang, H. Ahorn, M. Freissmuth, and C. Nanoff Binding of Calmodulin to the D2-Dopamine Receptor Reduces Receptor Signaling by Arresting the G Protein Activation Switch J. Biol. Chem., October 13, 2000; 275(42): 32672 - 32680. [Abstract] [Full Text] [PDF]

				O. Kudlacek, T. Mitterauer, C. Nanoff, M. Hohenegger, W.-J. Tang, M. Freissmuth, and C. Kleuss Inhibition of Adenylyl and Guanylyl Cyclase Isoforms by the Antiviral Drug Foscarnet J. Biol. Chem., January 26, 2001; 276(5): 3010 - 3016. [Abstract] [Full Text] [PDF]