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 beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion
proteins. Fusion
proteins provide a higher sensitivity for the analysis of beta(2)AR-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 > or =
UTP >
CTP >
ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(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 > or =
UTP > or =
CTP > or =
GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]
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 Galpha(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 approximately 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.