Showing papers on "GTP-Binding Protein alpha Subunits published in 2006"

Heterotrimeric G-proteins: a short history

Abstract: Some 865 genes in man encode G-protein-coupled receptors (GPCRs). The heterotrimeric guanine nucleotide-binding proteins (G-proteins) function to transduce signals from this vast panoply of receptors to effector systems including ion channels and enzymes that alter the rate of production, release or degradation of intracellular second messengers. However, it was not until the 1970s that the existence of such transducing proteins was even seriously suggested. Combinations of bacterial toxins that mediate their effects via covalent modification of the α-subunit of certain G-proteins and mutant cell lines that fail to generate cyclic AMP in response to agonists because they either fail to express or express a malfunctional G-protein allowed their identification and purification. Subsequent to initial cloning efforts, cloning by homology has defined the human G-proteins to derive from 35 genes, 16 encoding α-subunits, five β and 14 γ. All function as guanine nucleotide exchange on–off switches and are mechanistically similar to other proteins that are enzymic GTPases. Although not readily accepted initially, it is now well established that β/γ complexes mediate as least as many functions as the α-subunits. The generation of chimeras between different α-subunits defined the role of different sections of the primary/secondary sequence and crystal structures and cocrystals with interacting proteins have given detailed understanding of their molecular structure and basis of function. Finally, further modifications of such chimeras have generated a range of G-protein α-subunits with greater promiscuity to interact across GPCR classes and initiated the use of such modified G-proteins in drug discovery programmes.

Heterotrimeric G Proteins Facilitate Arabidopsis Resistance to Necrotrophic Pathogens and Are Involved in Jasmonate Signaling

Abstract: Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gbg dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Ga or Gb subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gb-deficient mutants while Ga-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gb-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gb-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Ga-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by Ga. We hypothesize that Gbg acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Inhibition of a background potassium channel by Gq protein α-subunits

Abstract: Two-pore-domain K(+) channels provide neuronal background currents that establish resting membrane potential and input resistance; their modulation provides a prevalent mechanism for regulating cellular excitability. The so-called TASK channel subunits (TASK-1 and TASK-3) are widely expressed, and they are robustly inhibited by receptors that signal through Galphaq family proteins. Here, we manipulated G protein expression and membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels in intact and cell-free systems to provide electrophysiological and biochemical evidence that inhibition of TASK channels by Galphaq-linked receptors proceeds unabated in the absence of phospholipase C (PLC) activity, and instead involves association of activated Galphaq subunits with the channels. Receptor-mediated inhibition of TASK channels was faster and less sensitive to a PLCbeta1-ct minigene construct than inhibition of PIP(2)-sensitive Kir3.4(S143T) homomeric channels that is known to be dependent on PLC. TASK channels were strongly inhibited by constitutively active Galphaq, even by a mutated version that is deficient in PLC activation. Receptor-mediated TASK channel inhibition required exogenous Galphaq expression in fibroblasts derived from Galphaq/11 knockout mice, but proceeded unabated in a cell line in which PIP(2) levels were reduced by regulated overexpression of a lipid phosphatase. Direct application of activated Galphaq, but not other G protein subunits, inhibited TASK channels in excised patches, and constitutively active Galphaq subunits were selectively coimmunoprecipitated with TASK channels. These data indicate that receptor-mediated TASK channel inhibition is independent of PIP(2) depletion, and they suggest a mechanism whereby channel modulation by Galphaq occurs through direct interaction with the ion channel or a closely associated intermediary.

Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors

Abstract: Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between α2-adrenergic receptors (α2R) and various combinations of Giα and Gβ subunit isoforms by measuring changes in FRET between Giα–yellow fluorescent protein and cyan fluorescent protein–Gβ chimeras in HeLa cells. All combinations of Giα1, -2, or -3 with Gβ1, -2, or -4 were activated to some degree by endogenous α2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Giα and Gβ subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of α2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express α2a- and α2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the α2aR subtype minimized the differences among coupling preferences for Giα and Gβ isoforms. The molecular properties of each Giα, Gβ, and α2-adrenergic receptor subtype influence signaling efficiency for the α2-adrenergic receptor-mediated signaling pathway.

Plant G protein heterotrimers require dual lipidation motifs of Gα and Gγ and do not dissociate upon activation

Abstract: In plants one bona fide Galpha subunit has been identified, as well as a single Gbeta and two Ggamma subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis Galpha subunit, GPalpha1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gbeta subunit, AGbeta1, and the Ggamma subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second Ggamma subunit, AGG2, did not require AGbeta1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer (FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AGbeta1 and AGG1 or AGbeta1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GPalpha1-AGbeta1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GPalpha1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GPalpha1 activation.

Structural and dynamical changes in an α-subunit of a heterotrimeric G protein along the activation pathway

Abstract: The Gα subunits of heterotrimeric G proteins (Gαβγ) mediate signal transduction via activation by receptors and subsequent interaction with downstream effectors. Crystal structures indicate that conformational changes in “switch” sequences of Gα, controlled by the identity of the bound nucleotide (GDP and GTP), modulate binding affinities to the Gβγ subunits, receptor, and effector proteins. To investigate the solution structure and dynamics of Gαi1 through the G protein cycle, nitroxide side chains (R1) were introduced at sites in switch II and at a site in helix α4, a putative effector binding region. In the inactive Gαi1(GDP) state, the EPR spectra are compatible with conformational polymorphism in switch II. Upon complex formation with Gβγ, motions of R1 are highly constrained, reflecting direct contact interactions at the Gαi1–Gβ interface; remarkably, the presence of R1 at the sites investigated does not substantially affect the binding affinity. Complex formation between the heterotrimer and activated rhodopsin leads to a dramatic change in R1 motion at residue 217 in the receptor-binding α2/β4 loop and smaller allosteric changes at the Gαi1–Gβγ interface distant from the receptor binding surface. Upon addition of GTPγS, the activated Gαi1(GTP) subunit dissociates from the complex, and switch II is transformed to a unique conformation similar to that in crystal structures but with a flexible backbone. A previously unreported activation-dependent change in α4, distant from the interaction surface, supports a role for this helix in effector binding.

A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins.

Abstract: The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.

Minimal determinants for binding activated G alpha from the structure of a G alpha(i1)-peptide dimer.

Abstract: G-proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G alpha subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP x AlF4(-)- and GTPgammaS-bound states of G alpha(i) subunits. KB-1753 blocks interaction of G alpha(transducin) with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G alpha in vitro. The crystal structure of KB-1753 bound to G alpha(i1) x GDP x AlF4(-) reveals binding to a conserved hydrophobic groove between switch II and alpha3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G alpha(i) subunits.

Compartmentalization of protein kinase A signaling by the heterotrimeric G protein Go

Abstract: Go, a member of the Go/i family, is the most abundant heterotrimeric G protein in brain. Most functions of Go are mediated by the Gβγ dimer; effector(s) for its α-subunit have not been clearly defined. Here we report that Goα interacts directly with cAMP-dependent protein kinase (PKA) through its GTPase domain. This interaction did not inhibit the kinase function of PKA but interfered with nuclear translocation of PKA while sparing its cytosolic function. This regulatory mechanism by which Go bifurcates PKA signaling may provide insights into how Go regulates complex processes such as neuritogenesis, synaptic plasticity, and cell transformation.

The molecular phylogeny of a nematode-specific clade of heterotrimeric G-protein alpha-subunit genes.

Abstract: In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein alpha subunit confers functional specificity to G-proteins. Vertebrate and insect Galpha-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Galpha genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Galpha subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Galpha gene classes, C. elegans and other nematodes also possess two lineage-specific Galpha gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Galpha genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution.