Distinct forms of the ß subunit of GTP-binding regulatory proteins identified by molecular cloning

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 2 subunits. The bovine transducin β subunit (β 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expreβed at lower levels than β 1 subunit mRNA in all tiβues examined. The β 1 and β 2 meβages are expreβed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26- kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP- ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min^-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist- induced, Ca(2+)-activated Cl- currents that were measured using a two- electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX- sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor- activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase

The effect of organic versus chemical fertilizers on insect pathogens

Insects such as the black cutworm are a major pest of seedling corn in Corn Belt states. Both the nematode Steinernema carpocapsae and the fungus Beauveria bassiana are potential biological control agents of this pest Because their survivability depends on moisture and they are affected adversely by nitrogen compounds, this project studied the effects of various fertilizers (fresh cow manure, composted cow manure, and urea) on these insect-killing organisms. Results showed that S. carpocapsae is more active in soils with no fertilizer amendment or with composted manure than with fresh manure or chemical fertilizer; B. bassiana is adversely affected by fresh manure. Investigation of factors that may enhance dispersal (which in turn may be a determining factor in the success of biological control) found that dispersal of two species of insect-killing nematodes was increased in the presence of earthworms

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phospho-transfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. N-15-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored (NH)-H-delta 1 tautomer of His-45, thereby rendering the N-epsilon 2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group

Caenorhabditis elegans Gα_q Regulates Egg-Laying Behavior via a PLCβ-Independent and Serotonin-Dependent Signaling Pathway and Likely Functions Both in the Nervous System and in Muscle

egl-30 encodes the single C. elegans ortholog of vertebrate Gα_q family members. We analyzed the expression pattern of EGL-30 and found that it is broadly expressed, with highest expression in the nervous system and in pharyngeal muscle. We isolated dominant, gain-of-function alleles of egl-30 as intragenic revertants of an egl-30reduction-of-function mutation. Using these gain-of-function mutants and existing reduction-of-function mutants, we examined the site and mode of action of EGL-30. On the basis of pharmacological analysis, it has been determined that egl-30 functions both in the nervous system and in the vulval muscles for egg-laying behavior. Genetic epistasis over mutations that eliminate detectable levels of serotonin reveals that egl-30requires serotonin to regulate egg laying. Furthermore, pharmacological response assays strongly suggest that EGL-30 may directly couple to a serotonin receptor to mediate egg laying. We also examined genetic interactions with mutations in the gene that encodes the single C. elegans homolog of PLCβ and mutations in genes that encode signaling molecules downstream of PLCβ. We conclude that PLCβ functions in parallel with egl-30 with respect to egg laying or is not the major effector of EGL-30. In contrast, PLCβ-mediated signaling is likely downstream of EGL-30 with respect to pharyngeal-pumping behavior. Our data indicate that there are multiple signaling pathways downstream of EGL-30 and that different pathways could predominate with respect to the regulation of different behaviors

Repetitive Segmental Structure of the Transducin β Subunit: Homology with the CDC4 Gene and Identification of Related mRNAs

Retinal transducin, a guanine nucleotide regulatory protein (referred to as a G protein) that activates a cGMP phosphodiesterase in photoreceptor cells, is comprised of three subunits. We have identified and analyzed cDNA clones of the bovine transducin β subunit that may be highly conserved or identical to that in other G proteins. From the cDNA nucleotide sequence of the entire coding region, the primary structure of a 340-amino acid protein was deduced. The encoded β subunit has a Mr of 37,375 and is comprised of repetitive homologous segments arranged in tandem. Furthermore, significant homology in primary structure and segmental sequence exists between the β subunit and the yeast CDC4 gene product. The Mr 37,375 β subunit polypeptide is encoded by a 2.9-kilobase (kb) mRNA. However, there exists in retina other β-related mRNAs that are divergent from the 2.9-kb mRNA on the basis of oligonucleotide and primer-extended probe hybridizations. All mammalian tissues and clonal cell lines that have been examined contain at least two β-related mRNAs, usually 1.8 and 2.9 kb in length. These results suggest that the mRNAs are the processed products of a small number of closely related genes or of a single highly complex β gene

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