2',3'-DIALDEHYDE GTP AS AN IRREVERSIBLE G-PROTEIN ANTAGONIST - DISRUPTION AND RECONSTITUTION OF G-PROTEIN-MEDIATED SIGNAL-TRANSDUCTION IN CELLS AND CELL-MEMBRANES
C. Nanoff et al., 2',3'-DIALDEHYDE GTP AS AN IRREVERSIBLE G-PROTEIN ANTAGONIST - DISRUPTION AND RECONSTITUTION OF G-PROTEIN-MEDIATED SIGNAL-TRANSDUCTION IN CELLS AND CELL-MEMBRANES, The Journal of biological chemistry, 269(50), 1994, pp. 31999-32007
The 2',3'-dialdehyde analogue of GTP, oGTP, was devised as an irrevers
ible antagonist of regulatory GTP-binding proteins (G proteins), Here,
we show that oGTP uncouples transmembrane signaling mediated by a set
of distinct G proteins both in isolated membranes and in whole cells,
In human platelet membranes, pretreatment with oGTP suppressed recept
or- and G protein-controlled regulation of adenylyl cyclase activity,
In chick neuronal cells, inhibition of the voltage-sensitive Ca2+-curr
ent by various membrane receptors (alpha(2)-adrenergic, somatostatin,
GABA(B)) was eliminated when oGTP was applied intracellularly in the w
hole cell patch-clamp configuration, Disruption of endogenous signalin
g pathways by oGTP occurred through specific blockage of the GTP-bindi
ng site of G protein alpha-subunits by the following criteria: (i) pre
treatment of membranes with oGTP blocked direct G protein activation b
y guanine nucleotides as well as labeling of G(s alpha) and G(i alpha)
, with the photoaffinity probe [(alpha-P-32]GTP azidoanilide, (ii) The
effect of oGTP was antagonized by the simultaneous introduction of gu
anosine 5'-(3-O-thio)triphosphate into the patch-clamped cell, (iii) T
he time to onset of action was similar for oGTP and guanosine 5'-O-thi
o)diphosphate, (iv) Inactivation of G protein-dependent signaling was
overcome by substituting G protein alpha-subunits. Addition of both th
e short and long form of recombinant G(s alpha)(rG(s alpha-s) and rG(s
alpha-L)) restored guanine nucleotide-dependent adenylyl cyclase acti
vity to oGTP-treated platelet membranes with rG(s alpha-L) being simil
ar to 3-10-fold more potent than rG(s alpha-s). This apparent preferen
ce was due to the intrinsically different activation rates of rG(s alp
ha-L) and rG(s alpha-s). When reconstituted with exogenous rG(s alpha)
, the A(2) adenosine receptor did not discriminate among the two forms
of rG(s alpha).Thus, G(s alpha-L) is the primary determinant of basal
cARIP formation in platelets, In contrast, neither the addition of va
rious recombinant subtypes of G(i/o) nor purified bovine brain py-dime
rs reconstituted adenylyl cyclase inhibition in oGTP-treated membranes
, All subtypes of G(i alpha) stimulated adenylyl cyclase, In the prese
nce of rG(s alpha), a conditional. stimulation by beta gamma-dimers wa
s observed, This pattern of stimulation shows that platelet adenylyl c
yclase is a type II-Iike isoform, Either a differently modified G prot
ein or an ancillary GTP-binding component is required for adenylyl cyc
lase inhibition in platelets, oGTP can be considered a useful tool for
disruption and reconstitution of transmembrane signaling mediated by
presumably all classes of heterotrimeric G proteins.