Lw. Runnels et Sf. Scarlata, Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors, BIOCHEM, 38(5), 1999, pp. 1488-1496
Phosphatidylinositide-specific phospholipase C-beta s play a key role in Ca
2+ signaling and are specifically activated by the cc, family of heterotrim
eric G proteins and as well as beta gamma subunits. We have determined the
affinity between G beta gamma subunits and GTP gamma S and CDP-liganded G a
lpha(q) subunits on membrane surfaces, and their respective affinities to P
LC-beta(1), -beta(2) and -beta(3) effecters by fluorescence spectroscopy. W
e find that activation of G alpha(q) by GTP gamma S decreases its affinity
for G beta gamma subunits at least 36-fold compared to the GDP-liganded for
m, but increases its affinity for PLC-beta s at least 40-200-fold depending
on the PLC-beta isoform. The affinity of G alpha(q)(GTP gamma S) is simila
r for PLC-beta(1) and -beta(3) and 10-fold stronger for PLC beta(2), which
corresponds to the reported relationship between the concentration of G alp
ha(q)(GTP gamma S) and PLC-beta activation on lipid bilayers. We find that
a large portion of the PLC-beta-G alpha(q), association energy lies within
the 400 residue C-terminal region of PLC-beta(1) since truncating this regi
on reduces its G alpha(q) affinity. In contrast, the isolated N-terminal re
gion does not interact with G alpha(q). G beta gamma subunits interact with
all three PLC-beta isotypes, but only showed strong binding to PLC-beta(2)
, and activation of the three PLC-beta s by G beta gamma subunits parallels
this behavior. We also tested the possibility that both G alpha(q) and G b
eta gamma can simultaneously bind PLC-beta(2). Our data argue against simul
taneous binding and show that G alpha(q) and G beta gamma independently reg
ulate this effector.