Interaction of the Xanthine Nucleotide Binding Goα Mutant with G Protein-coupled Receptors

We constructed a double mutant version of the α subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoαX). We investigated the interaction between GoαX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPγS for XDP in the GoαXβγ heterotrimer. Second, the GoαXβγ complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoαX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoαX, lacking a bound nucleotide and βγ, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoαX with receptor in COS-7 cells. The empty form of GoαX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoαX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that Goα X specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors

Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins

The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm

Archaeal ubiquity

In the seventeenth century, Antoine von Leeuwenhook used a simple microscope to discover that we live within a previously undetected microbial world containing an enormously diverse population of creatures. The late nineteenth and early twentieth century brought advances in microbial culture techniques and in biochemistry, uncovering the roles that microbes play in all aspects of our world, from causing disease to modulating geochemical cycles. In the last 25 years, molecular biology has revealed the complexity and pervasiveness of the microbial world and its importance for understanding the interactions that maintain living systems on the planet. The paper by Preston et al. (1) in this issue of the Proceedings provides a clear illustration of the power of these molecular techniques to describe new biological relationships and to pose important questions about the mechanisms that drive evolution. The analysis of ribosomal RNA gene sequences is one molecular approach that has radically altered our view of microbial diversity. Its application can be extended and expedited by the use of PCR. The confluence of these techniques has stimulated the rapid assembly of sequence information from homologues rRNA gene regions derived from virtually all classes of organisms. The data collected thus far support the scheme first presented by Woese et al. (2), which holds that the relationships among organisms can be summarized in the form of a universal phylogenetic tree comprised of one eukaryotic and two prokaryotic domains: the Eucarya, the Bacteria, and the Archaea (Fig. 1)

Activation of phospholipase C beta4 by heterotrimeric GTP-binding proteins

Transient transfection assays were used to determine how the activity of phospholipase C beta 4, which is preferentially expressed in retina, was regulated. An expression vector carrying the full-length cDNA corresponding to phospholipase C beta 4 was constructed and co- transfected into COS-7 cells together with cDNA encoding the alpha subunits of the Gq class and various beta and gamma subunits corresponding to the heterotrimeric GTP-binding proteins. We found that all the alpha subunits of the Gq class, including G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16 could activate PLC beta 4 and that none of the G beta gamma subunits that we tested including G beta 1 gamma 1, G beta 1 gamma 2, G beta 1 gamma 3, or G beta 2 gamma 2 activated phospholipase C beta 4. In control experiments, cotransfection with cDNA encoding the alpha subunit of transducin or Gi2 gave no activation of PLC beta 4. These results indicate that phospholipase C beta 4 is activated by G alpha subunits that are members of the Gq class, and, like the phospholipase C beta 1 isoform, it is refractory to activation in the transfection assay by many of the combinations of beta and gamma subunits found in the heterotrimeric G- proteins

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

We previously reported that the xanthine nucleotide binding Goα mutant, GoαX, inhibited the activation of Gi-coupled receptors. We constructed similar mutations in G11α and G16α and characterized their nucleotide binding and receptor interaction. First, we found that G11αX and G16αX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G11αX and G16αX interacted with βγ subunits in the presence of xanthine diphosphate. These experiments demonstrated that G11aαX and G16αX were xanthine nucleotide-binding proteins, similar to GoαX. Third, in COS-7 cells, both G11αX and G16αX inhibited the activation of Gq-coupled receptors, whereas only G16αX inhibited the activation of Gi-coupled receptors. Therefore, when in the nucleotide-free state, empty G11αX and G16αX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that GoαX, G11αX, and G16αX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G11αX and G16αX, but not GoαX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of Goα, G11α, and G16α can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors

Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins

Constitutively Active Galpha q and Galpha 13 Trigger Apoptosis through Different Pathways

We investigated the effect of expression of constitutively active Galpha mutants on cell survival. Transfection of constitutively active Galphaq and Galpha13 in two different cell lines caused condensation of genomic DNA and nuclear fragmentation. Endonuclease cleavage of genomic DNA was followed by labeling the DNA fragments and subsequent flow cytometric analysis. The observed cellular phenotype was identical to the phenotype displayed by cells undergoing apoptosis. To distinguish between the apoptosis-inducing ability of the two Galpha-subunits, the signaling pathways involved in this cellular function were investigated. Whereas Galpha q induced apoptosis via a protein kinaseC-dependent pathway, Galpha13 caused programmed cell death through a pathway involving the activation of the small G-protein Rho. Both of the pathways leading to apoptosis were blocked by overexpression of bcl-2. In contrast to other apoptosis-inducing systems, expression of constitutively active Galphaq and Galpha13 triggered apoptosis in high serum as well as in defined medium

Galpha 12 and Galpha 13 Are Phosphorylated during Platelet Activation

The ubiquitously expressed G-proteins G12 and G13 whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Galpha 12 and Galpha 13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alpha IIbbeta 3) activation. Phosphorylation of Galpha 12 and Galpha 13 could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in Galpha 12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha 12 or Galpha 13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C beta , delta , and epsilon were most effective in promoting phosphorylation of Galpha 12 and Galpha 13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha 12 and Galpha 13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C- terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha- subunits, G alpha q and/or G alpha 11