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We previously reported that the xanthine nucleotide binding G o ␣ mutant, G o ␣X, inhibited the activation of G i -coupled receptors. We constructed similar mutations in G 11 ␣ and G 16 Heterotrimeric G protein signaling pathways are commonly used to transduce signals across cell membranes in eukaryotic cells. G proteins contain three subunits, ␣, ␤, and ␥, and can be activated by hundreds of seven-transmembrane receptors. Binding of agonist to receptor activates the receptor, which then catalyzes the exchange of GTP for GDP bound to G protein ␣ subunits. Activated GTP-bound ␣ subunits and free ␤␥ subunits regulate a variety of cellular effectors, including enzymes and ion channels (1-3). G protein ␣ subunits can be divided into four families: G s , G i (G i , G o , and transducin), G q (G q , G 11 , G 14 , and G 16 ), and G 12 (G 12 and G 13 ). Some G protein-coupled receptors activate only one family of G proteins, whereas other receptors may activate multiple families of G proteins. G 16 and its mouse homologue G 15 behave promiscuously; they can be activated by all classes of G protein-coupled receptors (4).We recently reported that the xanthine nucleotide binding G o ␣ mutant, G o ␣X (a double mutant of G o ␣, D273N/Q205L) can interact with appropriate receptors on the membrane (5, 6). G o ␣X was regulated by xanthine nucleotides instead of guanine nucleotides. The empty form (nucleotide-free) of G o ␣X has been shown to form a stable complex with G o -coupled receptors and to inhibit the cognate receptor by competing with endogenous wild-type G proteins. In the present study, we investigated the functions of similar mutants in another G protein family. We found that both G 11 ␣X (G 11 ␣DN/QL) and G 16 ␣X (G 11 ␣DN/QL) were xanthine nucleotide-binding proteins. They bound XTP␥S, but not GTP␥S. These mutant proteins were also able to bind ␤␥ subunits only in the presence of XDP. In the nucleotide-free state, they interacted with their appropriate receptors and inhibited activation. Furthermore, G 11 ␣X and G 16 ␣X retained the same receptor binding specificity of the wild-type proteins. G 11 ␣X only inhibited G q -coupled receptors, but not G i -coupled receptors, whereas G 16 ␣X was able to inhibit receptors from both families. These results suggest that as with G o ␣X, G 11 ␣X, and G 16 ␣X can be used as dominant inhibitors against a subset of G protein-coupled receptors. Mutagenesis of G 11 ␣ and G 16 ␣-The D277N mutation was introduced in both wild-type G 11 ␣ and the activated mutant G 11 ␣Q209L. The D280N mutation was introduced in both wild-type G 16 ␣ and the activated G 16 ␣Q213L. The site-specific mutagenesis was conducted by polymerase chain reaction using oligonucleotide TTCCTCAACAAGAAG-GACCTTCTAGAAGAC for G 11 ␣ and TTTCTCAACAAAACCGACATC-CTGGAGGAGAAAATCCC for G 16 ␣. The cDNAs were subcloned into the pCIS vector under the control of a CMV promoter. EXPERIMENTAL PROCEDURES Materials-PurifiedExpression and Purification of His 6 -tagged G o ␣-Both wild-type G o ␣ and mutant G o ␣X were subcloned ...

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Go␣ mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Go␣ subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein Go␣D273N/Q205L (Go␣X) bound xanthine triphosphate, but not guanine triphosphate. Recombinant Go␣X (Go␣D273N/Q205L) formed heterotrimers with ␤␥ complexes only in the presence of xanthine diphosphate (XDP), 1 and the binding to ␤␥ was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the Go␣X protein underwent a conformational change similar to that of the activated wild-type Go␣. In transfected COS-7 cells, we demonstrate that the interaction between Go␣X and ␤␥ occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein ␣ subunit.G proteins transduce receptor-generated signals across the plasma membranes of eukaryotic cells. They are heterotrimeric complexes composed of ␣, ␤, and ␥ subunits. Each of the subunits belongs to a multigene protein family, containing at least 18 distinct ␣, 5 ␤, and 11 ␥ subunits. Hundreds of seventransmembrane receptors activated by a great variety of hormones, neuromediators, and growth factors are coupled to G proteins. Receptor-induced activation of a G protein leads to exchange of GDP for GTP bound to the ␣ subunit. The GTPbound ␣ subunit is released from the ␣␤␥ trimeric complex, and both free ␣ and ␤␥ dimers are capable of modulating activities of effector enzymes and ion channels (1-3). G protein-mediated signaling is complicated; a single receptor can activate more than one kind of heterotrimer, and both the activated ␣ and the ␤␥ subunits can interact with multiple effectors. For example, the thrombin receptor is known to couple to G 12 , Gi, and Gq family members (4), and physiological responses may be the result of contributions by both ␣ and ␤␥ subunits. Furthermore, cross-talk between these different G protein-regulated pathways makes the networks even more complex.One way to analyze this complex network is to specifically activate a particular G␣ in vivo to discern its function without interference from other G proteins. As a first step toward this goal, we used site-specific mutagenesis to switch the nucleotide specificity of G␣ from guanine to xanthine nucleotides. In cells, xanothine monophosphate is an intermediate in the biosynthesis of GMP; however, the steady-state concentrations of XDP and XTP are relatively low (5). Thus, by subsequent introduction of XTP, we should be able to specifically activate the mutant protein. Th...

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