title: Low-affinity interactions of BODIPY-FL-GTPgammaS and BODIPY-FL-GppNHp with G(i)- and G(s)-proteins.
abstract: 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 1100- to 5600-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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Kansas, Malott Hall, Room 5064, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gille
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D011485:Protein Binding; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: C486958:BODIPY FL GTPgammaS; C488296:BODIPY-FL-guanosine 5'-(beta,gamma-imido)triphosphate; D001896:Boron Compounds; D005456:Fluorescent Dyes; D013873:Thionucleotides; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs