Distinct interactions of GTP, UTP, and CTP with G_s proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G_s protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G_s activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the β₂-adreno-receptor (β₂AR) couples to G_s, and Sf9 insect cell membranes expressing β₂AR-Gα_s fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of β₂AR-G_s coupling than native systems. Nucleoside 5′-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP ≥ UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in β₂AR-Gα_s in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP ≥ UTP ≥ CTP ≥ GTP. NTPs inhibited β₂AR-Gα_s-catalyzed [γ-³²P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Gα_s than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G_s proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach ∼10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.