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Two Novel GPCR-Type G Proteins Are Abscisic Acid Receptors in Arabidopsis
Figure 7. Specific ABA Binding by GTG1
and GTG2
Each experiment was repeated five times and data
were averaged. Values are means ¡À SEM. Except
as described in (C), the reaction mix included 5 mM
GDP.
(A) Both GTG1 and GTG2 show saturable
ABA binding with increasing concentrations of
3H-ABA. No binding was detected when BSA
instead of GTG1 or GTG2 was used or by using
3H-benzoic acid (BnzA) instead of 3H-ABA. Inset
shows Scatchard plot analysis. Parameters
derived for GTG1 are KD = 35.8 ¡À 2.6 nM, with
R2 = 0.98 and for GTG2 are KD = 41.2 ¡À 3.1, with
R2 = 0.97.
(B) ABA binding by GTG1 and GTG2 is competed
by (¡À)-ABA but not by the biologically inactive
stereoisomer, (-)ABA. One hundred nanomolar
3H-ABA was used, with increasing concentrations
of cold ABA as indicated.
ÔÚÂÛÎÄÀïµÄÃèÊöÊÇ£ºThe G protein activity and GPCR-like
topology of GTG proteins along with
the significantly ABA hyposensitive
phenotypes of gtg1 gtg2 mutants led
to the intriguing possibility that the
GTG proteins constitute all or part of an
unusual class of G protein-coupled ABA
perception complex(es). To test this
possibility, we carried out direct ABAbinding
assays with 3H-ABA and purified
recombinant GTG proteins. Both GTG1
and GTG2 proteins showed saturable
and specific 3H-ABA binding, with
apparent KD (dissociation constant) values of 35.8 nM and
41.2 nM, respectively (Figure 7A). GTG proteins did not bind
3H-benzoic acid, another weak acid used as a negative control.
The ABA binding of GTG1 and GTG2 could be effectively
competed with unlabeled biologically active (¡À)-ABA, but not
by the biologically inactive ()-ABA isomer (Figure 7B). Following
the implication that the GTG proteins do not bind the ()-ABA
isomer, the above KD values would then be halved. No 3H-ABA
binding was observed when BSA or GPA1 was used as a control
for nonspecific binding (Figures 7A and 7C).
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