Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward ß-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

State-selective binding peptides for heterotrimeric G-protein subunits: novel tools for investigating G-protein signaling dynamics.

Heterotrimeric G-proteins, comprising Galpha, Gbeta, and Ggamma subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Galpha is complexed with the Gbetagamma dimer, and an active state in which GTP-bound Galpha is freed of its Gbetagamma binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise 'switch regions' of the Galpha subunit. Discrete differences in conformation between GDP- and GTP-bound Galpha underlie its nucleotide-dependent protein-protein interactions (e.g., with Gbetagamma/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Galpha (and free Gbetagamma) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Galpha and Gbetagamma signaling, and serving as biosensor tools in assays for Galpha activation including high throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.

Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C.

Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.

G16-mediated activation of nuclear factor kappaB by the adenosine A1 receptor involves c-Src, protein kinase C, and ERK signaling.

The G(i)-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-kappaB (NFkappaB). Here we demonstrate that an adenosine A1 agonist, N(6)-cyclohexyladenosine (CHA), activated IKKalpha/beta phosphorylation through PTX-insensitive G proteins in human lymphoblastoma Reh cells. To delineate the mechanism of action, different PTX-insensitive G proteins were expressed in human embryonic kidney 293 cells. Only Galpha(16) supported the CHA-induced IKK phosphorylation and NFkappaB-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. Gbetagamma subunits also modulated IKK/NFkappaB, as indicated by the stimulatory actions of Gbeta(1)gamma(2) and the abrogation of CHA-induced response by transducin. The participation of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/NFkappaB activation were demonstrated by employing specific inhibitors and dominant-negative mutants. Inhibition of c-Src and numerous intermediates along the extracellular signal-regulated (ERK) kinase cascade including Ras, Raf-1 kinase, and MEK1/2 abolished the CHA-induced IKK/NFkappaB activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NFkappaB regulation. Similar results were obtained using Reh cells. These data suggest that the G(16)-mediated activation of IKK/NFkappaB by CHA required a complex signaling network composed of multiple intermediates.

Stimulation of increases in intracellular calcium and prostaglandin E2 generation in Chinese hamster ovary cells expressing receptor-Galpha16 fusion proteins.

We examined whether fusion proteins of G protein-coupled receptors with the alpha subunit of G(16) (Galpha(16)) could activate downstream signals. We expressed fusion proteins of G(i)-coupled receptors, i.e. CX(3)C chemokine receptor 1 (CX(3)CR1) and M(2) receptor, in Chinese hamster ovary cells. An agonist for CX(3)CR1 induced greater increases in intracellular Ca(2+) and prostaglandin E(2) generation in cells expressing CX(3)CR1-Galpha(16) fusion protein than in cells expressing CX(3)CR1 alone or both CX(3)CR1 and Galpha(16) separately. Similarly, agonist-induced prostaglandin E(2) generation was greater in cells expressing M(2)-Galpha(16) fusion protein than ones expressing M(2) alone or both M(2) and Galpha(16) separately. In cells expressing fusion proteins with Galpha(16) of G(q)-coupled receptors, i.e. urotensin II receptor and M(1) receptor, the relevant agonists induced similar increases in intracellular Ca(2+) and prostaglandin E(2) generation as in ones expressing the receptor alone. In cells expressing urotensin II receptor-Galpha(16) fusion protein, prostaglandin E(2) generation exhibited a lower EC(50) value than the intracellular Ca(2+) increase. These results indicate that agonist-stimulated receptor-Galpha(16) fusion proteins are coupled to downstream signaling pathways, and suggest that receptor-Galpha(16) fusion proteins may be useful for screening for ligands of orphan G protein-coupled receptors and G(i)-coupled receptors.

Constitutively active Galpha16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism.

The hematopoietic-specific Galpha16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Galpha16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Galpha16 mutant, Galpha16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Galpha16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Galpha16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Galpha16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Galpha16. Demonstration of the involvement of different kinases in Galpha16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins.

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3.

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.

Distinct sites on G protein beta gamma subunits regulate different effector functions.

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents. Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K(+) channels. With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors. These data show that distinct domains within Gbetagamma subserve specific functional roles.

Molecular cloning and characterization of cDNAs encoding heterotrimeric G protein alpha and beta subunits from potato (Solanum tuberosum L.).

Two cDNAs, STGA2 and STGB2, that encode heterotrimeric G protein alpha and beta subunit proteins, respectively, were cloned from an early tuber cDNA library of potato (Solanum tuberosum cv. Superior). The cDNA of STGA2 encoded 384 amino acids, which showed 75-98% identities to plant Ga-subunits; STGB2 encoded 377 amino acids, which showed 83-92% identities to plant Gbeta-subunits. The transcript levels of the two genes were abundant in leaves, shoots, axially buds, unopened flowers, and active growing sprouts. However, the transcripts were barely detectable in roots. The expressions of STGA2 and STGB2 were up-regulated by light. Interestingly, the STGA2 and STGB2 gene expression showed synchronous patterns in the examined organs. During the early tuber development, the transcripts of STGA2 and STGB2 were abundant in unswollen stolons, swollen stolons, and new tubers, but were undetected in matured tubers. This indicates that potato Galpha- and beta-subunit genes are developmentally regulated. Based on these observations, we propose that heterotrimeric G proteins may be involved in the signaling pathway during potato tuber development.

Gbeta residues that do not interact with Galpha underlie agonist-independent activity of K+ channels.

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.

Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13.

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin.

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The alpha-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel alpha-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.