Low-affinity interactions of BODIPY-FL-GTPγS and BODIPY-FL-GppNHp with Gi- and Gs-proteins

BODIPY-FL-guanosine 5'-[gamma-thio]triphosphate (B-GTPgammaS) and BODIPY-FL-guanosine 5'-[beta,gamma-imido]triphosphate (B-GppNHp) induce fluorescence changes upon binding to purified G(s)/G(i)-proteins and were suggested to serve as probes for monitoring receptor-mediated G-protein activation. However, B-GTPgammaS and B-GppNHp bound to receptor-Galpha(s)/Galpha(i) fusion proteins expressed in Sf9 cell membranes with 1,100- to 5,600-fold- and 17- to 55-fold lower affinity than GTPgammaS and GppNHp, respectively. The affinity of B-GTPgammaS/B-GppNHp for G(s)/G(i)-proteins was considerably lower than the affinity of N-methylanthraniloyl (MANT)-substituted GTP analogs for G(s)/G(i)-proteins. B-GTPgammaS/B-GppNHp were much less potent than GTPgammaS/GppNHp at regulating adenylyl cyclase (AC) via G(s)- and G(i)-proteins. B-GTPgammaS/B-GppNHp were similarly efficient as GTPgammaS/GppNHp at activating G(i), but less efficient at activating G(s). In contrast to MANT-GTPgammaS/MANT-GppNHp, B-GTPgammaS/B-GppNHp were inefficient at directly inhibiting AC. In conclusion, the bulky BODIPY group strongly reduces the affinity of GTPgammaS/GppNHp for G-proteins, limiting the use of B-GTPgammaS/B-GppNHp as fluorescence probes.