Functionally nonequivalent interactions of guanosine 5'-triphosphate, inosine 5'-triphosphate, and xanthosine 5'-triphosphate with the retinal G-protein, transducin, and with Gi-proteins in HL-60 leukemia cell membranes.

G-proteins mediate signal transfer from receptors to effector systems. In their guanosine 5'-triphosphate (GTP)-bound form, G-protein alpha-subunits activate effector systems. Termination of G-protein activation is achieved by the high-affinity GTPase [E.C. 3.6.1.-] of their alpha-subunits. Like GTP, inosine 5'-triphosphate (ITP) and xanthosine 5'-triphosphate (XTP) can support effector system activation. We studied the interactions of GTP, ITP, and XTP with the retinal G-protein, transducin (TD), and with G-proteins in HL-60 leukemia cell membranes. TD hydrolyzed nucleoside 5'-triphosphates (NTPs) in the order of efficacy GTP > ITP > XTP. NTPs eluted TD from rod outer segment disk membranes in the same order of efficacy. ITP and XTP competitively inhibited TD-catalyzed GTP hydrolysis. In HL-60 membranes, the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and leukotriene B4 (LTB4) effectively activated GTP and ITP hydrolysis by Gi-proteins. fMLP and LTB4 were at least 10-fold more potent activators of ITPase than of GTPase. Complement C5a effectively activated the GTPase of Gi-proteins but was only a weak stimulator of ITPase. The potency of C5a to activate GTP and ITP hydrolysis was similar. The fMLP-stimulated GTPase had a lower Km value than the fMLP-stimulated ITPase, whereas the opposite was true for the Vmax values. fMLP, C5a, and LTB4 did not stimulate XTP hydrolysis. Collectively, our data show that GTP, ITP, and XTP bind to G-proteins with different affinities, that G-proteins hydrolyze NTPs with different efficacies, and that chemoattractants stimulate GTP and ITP hydrolysis by Gi-proteins in a receptor-specific manner. On the basis of our results and the data in the literature, we put forward the hypothesis that GTP, ITP, and XTP act as differential signal amplifiers and signal sorters at the G-protein level.