Characterization of a Goα Mutant That Binds Xanthine Nucleotides

Abstract

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...

Inhibition of Subsets of G Protein-coupled Receptors by Empty Mutants of G Protein α Subunits in Go, G11, and G16

“…We also showed that the mutant proteins of G 11 ␣X and G 16 ␣X expressed in COS-7 cells interacted with ␤␥ subunits in a XDP-dependent fashion; they only bound ␤␥ when XDP was available, whereas G 11 ␣DN and G 16 ␣DN did not. These results are consistent with previous findings using G o ␣X and G o ␣DN; the single DN mutation resulted in a loss of ability to bind nucleotides, whereas the double DN/QL mutation lead to xanthine nucleotide binding (5). Although the mutation of Asp 3 Asn in the conserved NKXD motif of G protein ␣ subunits was expected to switch the nucleotide specificity of the mutated protein from guanine nucleotide to xanthine nucleotide, according to the available crystal structures of G protein ␣ subunits and other GTP-binding proteins, we observed that the single DN mutation resulted in a protein not able to bind either nucleotides in three G protein ␣ subunits: G o ␣, G 11 ␣, and G 16 ␣.…”

“…Section 1734 solely to indicate this fact. (5,6). 0.5 g of purified G o ␣ was first incubated with 1 g of transducin ␤␥ and 100 g of m2 MAChR membrane in TED buffer with 10 M GDP, 0.1 mM MgCl 2 , and 1 M ATP for 0.5 h. The reaction was started with the addition of 0.1 M GTP␥S (20,000 cpm/pmol) and 100 M carbachol.…”

“…Expression and Purification of His 6 -tagged G o ␣-Both wild-type G o ␣ and mutant G o ␣X were subcloned into the Escherichia coli expression vector pET-15b (Novagen) with a His 6 tag at the N terminus (5). The recombinant proteins were expressed and purified as described previously.…”

“…Permeabilization of COS-7 Cell Membranes-Membranes of transfected COS-7 cells were permeabilized as described (5). Cells were washed and incubated in 200 ml of permeabilization solution consisting of 115 mM KCl, 15 mM NaCl, 0.5 mM MgCl 2 , 20 mM Hepes-NaOH, pH 7, 1 mM EGTA, 100 mM ATP, 0.37 mM CaCl 2 (to give a free Ca 2ϩ concentration of 100 nM), and 200 units/ml ␣-toxin with or without 0.1 mM XDP for 10 min at 37°C.…”

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

“…For several classes of GTP-binding proteins, a strategy was devised where an aspartate to asparagine mutation shifted nucleotide specificity from guanine to xanthine ( Figure 1B). Examples include H-Ras (Zhong et al, 1995), EF-Tu (Hwang and Miller, 1987;Weijland et al, 1993), Ypt1 (Jones et al, 1995), Rab-5 (Hoffenberg et al, 1995;Rybin et al, 1996), FtsY (Powers and Walter, 1995), adenylosuccinate synthetase (Kang et al, 1994) and Goa (although here a second mutation was essential for shifting nucleotide specificity) (Yu et al, 1997). Xanthine nucleotides are only transiently produced during purine metabolism and are not populated as triphosphates in vivo, allowing for separation of the signaling effect of a specific GTPase in the background of other GTPases.…”