THIOPHOSPHORYLATION OF THE G-PROTEIN BETA-SUBUNIT IN HUMAN PLATELET MEMBRANES - EVIDENCE AGAINST A DIRECT PHOSPHATE TRANSFER-REACTION TO G(ALPHA)SUBUNITS
M. Hohenegger et al., THIOPHOSPHORYLATION OF THE G-PROTEIN BETA-SUBUNIT IN HUMAN PLATELET MEMBRANES - EVIDENCE AGAINST A DIRECT PHOSPHATE TRANSFER-REACTION TO G(ALPHA)SUBUNITS, Molecular pharmacology, 49(1), 1996, pp. 73-80
A direct phosphate transfer reaction from the G protein beta subunits
to either G(s alpha) or G(i alpha) has been proposed to account for th
e ability of thiophosphorylated transducin beta gamma-dimers to bidire
ctionally regulate adenylyl cyclase activity in human platelet membran
es. We searched for experimental evidence for this reaction. Incubatio
n of human platelet membranes with [S-35]guanosine-5'-(3-O-thio)tripho
sphate ([S-35]GTP gamma S) results in the predominant incorporation of
[S-35]thiophosphate into a 36-kDa protein, which comigrates with the
G protein beta subunit and is immunoprecipitated by a beta subunit-spe
cific antiserum. Thiophosphorylation of the beta subunit is specific f
or guanine nucleotides and abolished by the histidine-modifying agent
diethylpyrocarbonate and heat and acid treatment. Dephosphorylation of
[S-35]thiophosphorylated beta subunits is accelerated in the presence
of GDP, but not ADP, UDP, or guanosine-5'-(2-O-thio)diphosphate. Neit
her the thiophosphorylation nor the dephosphorylation is sensitive to
receptor agonists (alpha(2)-adrenergic, A(2) adenosine, thrombin, or i
nsulin), and purified G protein alpha subunits do not act as thiophosp
hate donors. An approach was designed to demonstrate direct thiophosph
ate transfer to protein-bound nucleotides; platelet membranes were seq
uentially 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 [S-35]GTP gamma S. This reaction is specifi
c because both oxidation and reduction are required and pretreatment o
f platelet membranes with 2',3'-dialdehyde GTP gamma S or diethylpyroc
arbonate blocks the subsequent labeling in oxidized and reduced membra
nes. The G protein beta subunit may participate in this thiophosphate
transfer reaction. Most important, however, no labeled G protein alpha
subunits (G(s alpha)) and G(i alpha) were recovered by immunoprecipit
ation from oxidized and reduced membranes subsequent to the addition o
f [S-35]GTP gamma S. Thus,our results clearly rule out the existence o
f a postulated G protein activation by phosphate transfer reactions, w
hich lead to the formation of GTP from GDP prebound to the or subunit.