Functional Analysis of a Dominant Negative Mutant of Gαi2

The key event in receptor-catalyzed activation of heterotrimeric G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta complexes. We have previously identified a mutation that abolished GTP binding in Galpha(o) (S47C) and demonstrated that the mutant retained the ability to bind beta and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V. Z., Quick, M. W., Aragay, A. M., Davidson, N., Lester, H. A., and Simon, M. I.(1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of Galpha (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the Galpha S48C mutant prevented stimulation of phospholipase C (PLC) beta2 by free beta subunit complexes. This effect of Galpha(i) S48C was not readily reversible in contrast to the inhibitory effect of wild-type Galpha, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta from the G protein heterotrimer. Coexpression of Galpha(i) S48C or the wild-type Galpha also dramatically decreased G-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and Galpha. Activation of PLC via endogenous G(q) or G in the presence of alpha1C adrenergic receptors was similarly attenuated by coexpression of Galpha(i) or Galpha(i) S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type Galpha(i) subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type Galpha(i) inhibitory effect by pertussis toxin can be explained by stabilization of Galpha(i) binding to beta as a result of ADP-ribosylation, while Galpha(i) S48C mutant binds beta irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the Galpha and G-mediated activation of PLC by cotransfected Galpha(i) is the competition between Galpha(i) and Galpha or G for the beta complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta in the integration of signals controlling phosphoinositide release through different Galpha families

Gα16, a G Protein α Subunit Specifically Expressed in Hematopoietic Cells

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein α subunit, Gα16, is specifically expressed in human cells of the hematopoietic lineage. The Gα16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the Gq class of α subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein α subunits, the Gα16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the Gα16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. Gα16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of Gα16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress Gα16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that Gα16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin

The N terminus of phosducin is involved in binding of βγ subunits of G protein

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the βγ subunits of guanine nucleotide-binding proteins (G proteins) (Gβγ) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind Gβγ was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit Gβγ-mediated phospholipase C β_2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-Gβγ interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to Gβγ. In contrast, the C-terminal half failed to bind to Gβγ. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between Gβγ and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residu

Characterization of a Goα Mutant That Binds Xanthine Nucleotides

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 Goα mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goα subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoαD273N/Q205L (GoαX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoαX (GoαD273N/Q205L) formed heterotrimers with βγ complexes only in the presence of xanthine diphosphate (XDP), and the binding to βγ was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoαX protein underwent a conformational change similar to that of the activated wild-type Goα. In transfected COS-7 cells, we demonstrate that the interaction between GoαX and βγ 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 α subunit

Characterization of G-protein α subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines

Murine Gα14 and Gα15 cDNAs encode distinct α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins). These alpha subunits are related to members of the Gq class and share certain sequence characteristics with Gαq, Gα11, and Gα16, such as the absence of a pertussis toxin ADP-ribosylation site. Gα11 and Gαq are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas Gα15 is primarily restricted to hematopoietic lineages. Among hematopoietic cell lines, Gα11 mRNA is found in all cell lines tested, Gαq is expressed widely but is not found in most T-cell lines, Gα15 is predominantly expressed in myeloid and B-cell lineages, and Gα14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells. Polyclonal antisera produced from synthetic peptides that correspond to two regions of Gα15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with Gα15 cDNA. The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function

Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding

Nucleotide binding properties of the G protein ɑ subunit G_oɑ were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [^(35)S]GTPyS (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G_oɑ cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G_oɑ for GTPyS or Mg^(2+) were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21^(ras) proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPyS binding but retained the capacity to interact with the βy subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G_oɑ increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl^- response, suggesting that this mutation resulted in a dominant negative phenotype

Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1β, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1β were detected within 6 h and peaked at 12–24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1−/−Casp4(11)del, Panx1−/− and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas

Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies

Application of surface plasmon resonance for analysis of protein–protein interactions in the G protein‐mediated signal transduction pathway

Hundreds of extracellular stimuli are received by cells via the pathways consisting of three basic components: cell‐surface receptors, heterotrimeric G proteins, and intracellular effector enzymes or ion channels. A number of additional molecules, including G protein‐coupled receptor kinases (GRKs), phosducin and Ca2+‐binding proteins modulate signal transduction through these cascades. Understanding how these universal pathways work requires a detailed analysis of the interactions between these proteins. The recently emerged technology of surface plasmon resonance (SPR) can study protein–protein interactions by measuring not only the equilibrium binding constants, but also the association and dissociation rates. This article reviews experimental design used by researchers to analyze different components of the G protein pathway by SPR and focuses on the insights this technique provides regarding the kinetics, structure–function aspects and regulation of specific molecular events in the cascade. Copyright © 2000 John Wiley & Sons, Ltd

A Gs-RhoGEF interaction: An old G protein finds a new job

The heterotrimeric G proteins are known to have a variety of downstream effectors, but G s was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated G s can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by G s -coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gα s subfamily regulates activity of Rho GTPases extends our understanding of Gα s activity and establishes RhoGEF coupling as a universal Gα function