The C-terminus of the G protein α subunit controls the affinity of nucleotides

The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.     

Characterization of the putative α subunit of a heterotrimeric G protein in rice

A recombinant protein with a cDNA that encodes the putative subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice ) with an apparent molecular mass of 45 kDa was bound with guanosine 5-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice . A 45 kDa polypeptide in the plasma membrane, as well as rGrice , was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the subunits of heterotrimeric G proteins in animals, suggesting that the putative subunit is truly the subunit itself.     

Expression profile of the α subunit of the heterotrimeric G protein in rice

Previous studies on the activity of the rice Gα promoter using a β-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.Key words: expression pattern, Gα subunit, GUS staining pattern, heterotrimeric G protein, riceThe rice mutant d1 is deficient in the heterotrimeric G protein α subunit (Gα). Recently it was found that the dwarfism phenotype of d1 is due to a reduction in cell numbers.¹ This discovery has led to new questions regarding how rice Gα regulates cell number, and which other signaling molecules are involved in this process in various tissues and at different development stages. Studies of d1 suggest that rice Gα participates in both gibberellin signaling^2–4 and brassinosteroid signaling.^5–8 Promoter studies using the β-Glucuronidase (GUS) reporter indicate that Gα expression is highest in developing organs.¹ In this paper, we report on the expression pattern of a Gα promoter::GUS construct in seeds and developing leaves of rice.     

Function of α subunit of heterotrimeric G protein in brassinosteroid response of rice plants

The α subunit of heterotrimeric G-proteins (Gα) is involved in a broad range of aspects of the brassinosteroid (BR) response, such as the enhancement of lamina bending. However, it has been suggested from epistatic analysis of d1 and d61, which are mutants deficient for Gα and the BR receptor BRI1, that Gα and BRI1 may function via distinct pathways in many cases. In this study, we investigated further the genetic interaction between Gα and BRI1. We report the analysis of transformants of T65d1 and T65d1/d61-7 into which were introduced a constitutively active form of Gα, Q223L. The application of 24-epi-brassinolide (24-epiBL) to T65d1 expressing Q223L still resulted in elongation of the coleoptile and, in fact, it was enhanced over the wild-type plant (WT) level in a concentration dependent manner. In T65d1/d61-7 expressing Q223L, the seed size was enlarged over that of d61-7 due to activation of Gα. These results suggest that Q223L is able to augment the BR response in response to 24-epiBL and also that Q223L functions independently of BRI1 in the process of determining seed morphology, given that Q223L was functional in the BRI1-deficient mutant, d61-7.Key words: brassinosteroid, BRASSINOSTEROID INSENSITIVE1 (BRI1), genetic interaction, G-protein α subunit, rice plants, seed morphology, transgenic plants     

Mating systems and protein–protein interactions determine evolutionary rates of primate sperm proteins

To assess the relative impact of functional constraint and post-mating sexual selection on sequence evolution of reproductive proteins, we examined 169 primate sperm proteins. In order to recognize potential genome-wide trends, we additionally analysed a sample of altogether 318 non-reproductive (brain and postsynaptic) proteins. Based on cDNAs of eight primate species (Anthropoidea), we observed that pre-mating sperm proteins engaged in sperm composition and assembly show significantly lower incidence of site-specific positive selection and overall lower non-synonymous to synonymous substitution rates (d_N/d_S) across sites as compared with post-mating sperm proteins involved in capacitation, hyperactivation, acrosome reaction and fertilization. Moreover, database screening revealed overall more intracellular protein interaction partners in pre-mating than in post-mating sperm proteins. Finally, post-mating sperm proteins evolved at significantly higher evolutionary rates than pre-mating sperm and non-reproductive proteins on the branches to multi-male breeding species, while no such increase was observed on the branches to unimale and monogamous species. We conclude that less protein–protein interactions of post-mating sperm proteins account for lowered functional constraint, allowing for stronger impact of post-mating sexual selection, while the opposite holds true for pre-mating sperm proteins. This pattern is particularly strong in multi-male breeding species showing high female promiscuity.     

Isolation and characterization of gene encoding G protein α subunit protein responsive to plant hormones and abiotic stresses in Brassica napus

G protein plays an important role in signal pathways and involved in various signal transduction systems in plant. A full-length cDNA encoding a putative G protein α subunit (Gα), designated as BnGA1, was isolated from Brassica napus. The expression of BnGA1 in different B. napus tissues and developmental stags was analyzed using real-time PCR. The results showed that BnGA1 expressed was high in root, cotyledon and shoot apex. Stage expression pattern analysis revealed that BnGA1 expressed strongly at the 7th day, the bolting stage and fruiting stage. In addition, the expression of BnGA1 was analyzed under different concentrations of four plant hormones. The expression of BnGA1 was significantly induced by the high concentrations of abscisic acid (ABA) and brassinosteroid (BR). The expression of BnGA1 was also induced by low gibberellins acid 3 (GA₃) concentrations and higher GA₃ concentrations inhibit the expression of BnGA1. However, the expression of BnGA1 did not significantly regulated by exogenous indole-3-acetic acid (IAA). Moreover, the expression of BnGA1 under different abiotic stresses was analyzed at different time points. The BnGA1 was up-regulated in salt and drought stress and down-regulated in heat and cold stress. These expression results suggested that BnGA1 play an important role in plant hormones signal pathways and BnGA1 may be involved in plant defense system against environmental stresses in B. napus.     

Characterization of a G protein α subunit encoded gene from the dimorphic fungus-Tremella fuciformis

Antonie van Leeuwenhoek - Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries...     

Identification of heterotrimeric G protein α and β subunits in rice

Like those in mammals, heterotrimeric G protein complexes have been implicated in signal transduction pathways in plants; however, the subunits themselves have not been isolated. In this study, the rice heterotrimeric G protein subunits α (Gα) and β (Gβ) were purified by affinity chromatography using anti-Gα and -Gβ antibodies and SDS-PAGE. Six and seven peptides, respectively, were identified by mass spectrometry and identified as the translation products of the Gα gene RGA1 and Gβ gene RGB1. During purification, the subunits dissociated easily from the G protein complex.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Expression and subcellular distribution of UNC119a,a protein partner of transducin α subunit in rod photoreceptors

A recently discovered interaction of rod transducin α subunit (Gα_t1) with UNC119a is thought to be important for transducin trafficking in photoreceptors. In this study, we analyzed the subcellular distribution of UNC119a under different conditions of illumination in vivo. Analyses by immunofluorescence and Western blotting of retina serial tangential sections demonstrated that UNC119a resides predominantly in the rod inner segment, with a small fraction of UNC119a also appearing to infiltrate the rod outer segment. Such a distribution is consistent with the proposed role of UNC119a in facilitating transducin transport from the rod inner segment to the outer segment in the dark. In addition, UNC119a was present in smaller amounts in the cell body and synaptic region of rods. The profile of UNC119a subcellular distribution remained largely unchanged under all tested conditions of illumination, and correlated with the profile of Gα_t1 following its light-dependent translocation. Quantification by Western blotting suggested that mouse retina contains ~ 17 pmol of UNC119a, giving a ~ 1 to 4 molar ratio of UNC119a to Gα_t1. Hence, light-translocated Gα_t1 can serve as a major partner of UNC119a. Supporting this role, the levels of UNC119a were downregulated by about 2-fold in mouse retina lacking Gα_t1. As a dominant partner, Gα_t1 may potentially modulate the function of other known UNC119a-interacting proteins involved in photoreceptor ciliary trafficking and synaptic regulation, in a light-dependent manner.     

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.     

Molecular cloning and characterization of RGA1 encoding a G protein α subunit from rice (Oryza sativa L. IR-36)

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library prepared from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 M [adenylate-³²P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.     

Signaling by a non-dissociated complex of G protein βγ and α subunits stimulated by a receptor-independent activator of G protein signaling, AGS8

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.     

Mutational analysis of splicing activities of ribonucleotide reductase α subunit protein from lytic bacteriophage P1201

A CP1201 RIR1 intein is found in the ribonucleotide reductase alpha subunit (RNR α subunit) protein of lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078. This intein can be over-expressed and spliced in Escherichia coli NovaBlue cells. Mutations of C539, the N-terminal residue of the C-extein in the CP1201 RIR1 protein, led to the changes of pattern and level of protein-splicing activities. A G392S variant was found to be a temperature-sensitive protein with complete splicing activity at 17 and 28°C but not at 37°C or higher. We also found that the cleavage at the CP1201 RIR1 intein C-terminus of the double mutant G392S/C539G was blocked, but other cleavage activities could be efficiently performed at 17°C. G392S/C539G variant possessed the properties of low-temperature-induced cleavage at the intein N-terminus.     

Syntenic mapping and chromosomal localization of bovine α and β interferon genes

The previous assignment of bovine -(IFNA) and -(IFNB) interferon gene families to syntenic group U18 was confirmed with additional cDNA probes and a bovine-rodent hybrid somatic cell panel representing all 29 bovine autosomal syntenic groups. Fluorescent in situ hybridization (FISH) localized these genes to bovine Chromosome (Chr) 8 band 15 and demonstrates that with biotinylated plasmids, as few as five tandemly arrayed sequences can be detected by conventional fluorescent microscopy. This technique can be applied to physical mapping of other multicopy genes in domestic animals.     

Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis

During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.     

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.     

A constitutively active Gα subunit provides insights into the mechanism of G protein activation

The activation of Gα subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of Gα mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at position 56 within the linker region connecting the helical and GTP-binding domains of a Gα chimera, designated αT*, yields a more readily exchangeable state for guanine nucleotides. Here we show that GDP-GTP exchange on αT*(G56P), in the presence of the light-activated GPCR, rhodopsin (R*), is less sensitive to the β1γ1 subunit complex than to wild-type αT*. We determined the X-ray crystal structure for the αT*(G56P) mutant and found that the G56P substitution leads to concerted changes that are transmitted to the conformationally sensitive switch regions, the α4-β6 loop, and the β6 strand. The α4-β6 loop has been proposed to be a GPCR contact site that signals to the TCAT motif and weakens the binding of the guanine ring of GDP, whereas the switch regions are the contact sites for the β1γ1 complex. Collectively, these biochemical and structural data lead us to suggest that αT*(G56P) may be adopting a conformation that is normally induced within Gα subunits by the combined actions of a GPCR and a Gβγ subunit complex during the G protein activation event.     

Regional chromosomal assignment of genes encoding the α and β subunits of human complement protein C8 to 1p32

Summary The genes encoding the and subunits of human complement protein C8 previously mapped to chromosome 1 have been further localised to 1p32 by in situ hybridisation using biotinylated 2.4-kb human cDNA clones encoding the and subunits of human complement protein C8 as probes.