Big G,little G: G proteins and actin cytoskeletal reorganization

It has been known for some time that stimulation of heterotrimeric G protein-coupled receptors can cause cytoskeletal reorganization. A recent report in Developmental Cell demonstrates that the activation of a tyrosine kinase, C-terminal Src kinase, by heterotrimeric G protein subunits provides the trigger for Rho-dependent actin stress fiber formation.     

A dimeric RNA quadruplex architecture comprised of two G:G(:A):G:G(:A) hexads,G:G:G:G tetrads and UUUU loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.     

Two G12 family G proteins, G alpha12 and G alpha13, show different subcellular localization

The G alpha subunits of the G12 family of heterotrimeric G proteins, G alpha12 and G alpha13, are closely related in sequences and some effectors, but they often act through different pathways or bind to different proteins. We have examined subcellular distribution of these two G proteins and found that endogenous G alpha12 and G alpha13 localize in membrane and cytoplasmic fractions, respectively. Exogenously expressed G alpha12 and G alpha13 also localize in membrane and cytoplasmic fractions, respectively, in COS-7 cells. Stimulation of lysophosphatidic acid receptor coupled to G alpha13 markedly promotes the translocation of G alpha13 from cytoplasm to membrane. This different localization of G alpha12 and G alpha13 may explain some of the nonoverlapping actions of G alpha12 and G alpha13.     

Signalling mechanisms of RhoGTPase regulation by the heterotrimeric G proteins G12 and G13

G protein-mediated signal transduction can transduce signals from a large variety of extracellular stimuli into cells and is the most widely used mechanism for cell communication at the membrane. The RhoGTPase family has been well established as key regulators of cell growth, differentiation and cell shape changes. Among G protein-mediated signal transduction, G12/13-mediated signalling is one mechanism to regulate RhoGTPase activity in response to extracellular stimuli. The alpha subunits of G12 or G13 have been shown to interact with members of the RH domain containing guanine nucleotide exchange factors for Rho (RH-RhoGEF) family of proteins to directly connect G protein-mediated signalling and RhoGTPase signalling. The G12/13-RH-RhoGEF signalling mechanism is well conserved over species and is involved in critical steps for cell physiology and disease conditions, including embryonic development, oncogenesis and cancer metastasis. In this review, we will summarize current progress on this important signalling mechanism.     

G1 and G2 chalones of the gastric mucosa

A study was made of the action of human gastric mucosa G1 and G2 chalones on cellular regeneration of mouse gastric mucosa and of the duration of their maximal effect. Chalone fractions were obtained from the mucous membranes of 21 stomachs resected for peptic ulcer by the method of fractional ethanol precipitation. The data indicate that the maximal inhibitory action of G1 chalone occurs in 3, whereas that of chalone G2 in 6 hours. Some specificity of the action of chalones was discovered depending on the part of the gastric mucosa from which they were obtained.     

G proteins G12 and G13 control the dynamic turnover of growth factor-induced dorsal ruffles

Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble ( approximately 20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G(12) and G(13) control the rate of disassembly of dorsal ruffles. In Galpha(12)(-/-)Galpha(13)(-/-) fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer ( approximately 60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without Galpha(12) and Galpha(13). Thus, Galpha(12)/Galpha(13) critically regulate dorsal ruffle turnover.     

Antagonistic regulation of neurite morphology through Gq/G11 and G12/G13

The induction of neurite retraction and growth cone collapse via G-protein-coupled receptors is involved in developmental as well as regenerative processes. The role of individual G-protein-mediated signaling processes in the regulation of neurite morphology is still incompletely understood. Using primary neurons from brains lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13), we show here that G(12)/G(13)-mediated signaling is absolutely required for neurite retraction and growth cone collapse induced by the blood-borne factors lysophosphatidic acid and thrombin. Interestingly, the effects of lysophosphatidic acid were mediated mainly by G(13), whereas thrombin effects required G(12). Surprisingly, lack of Galpha(q)/Galpha(11) resulted in overshooting responses to both stimuli, indicating that G(q)/G(11)-mediated signaling most likely via activation of Rac antagonizes the effects of G(12)/G(13).     

Micropropagation of four Gentiana species (G. lutea, G. cruciata, G. purpurea and G. acaulis)

The growth of axillary shoots was initiated on nodal stem segments, excised from aseptically grown seedlings of Gentiana acaulis L., G. cruciata L., G. lutea L. and G. purpurea L. In later subcultures, a basal callus tissue developed on the shoots, giving rise to de novo formed buds. Optimum benzyladenine and indoleacetic acid combinations for shoot development were established. They were slightly different in the four species. From 35-70% of shoots rooted spontaneously, except in G. lutea, in which adventitious roots were induced by applying naphthaleneacetic acid. It was conduded that the four Gentiana species were amenable to propagation in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.     

A dimeric DNA interface stabilized by stacked A.(G.G.G.G).A hexads and coordinated monovalent cations

We report on the identification of an A.(G.G.G.G).A hexad pairing alignment which involves recognition of the exposed minor groove of opposing guanines within a G.G.G.G tetrad through sheared G.A mismatch formation. This unexpected hexad pairing alignment was identified for the d(G-G-A-G-G-A-G) sequence in 150 mM Na(+) (or K(+)) cation solution where four symmetry-related strands align into a novel dimeric motif. Each symmetric half of the dimeric "hexad" motif is composed of two strands and contains a stacked array of an A.(G.G.G.G).A hexad, a G.G.G.G tetrad, and an A.A mismatch. Each strand in the hexad motif contains two successive turns, that together define an S-shaped double chain reversal fold, which connects the two G-G steps aligned parallel to each other along adjacent edges of the quadruplex. Our studies also establish a novel structural transition for the d(G-G-A-G-G-A-N) sequence, N=T and G, from an "arrowhead" motif stabilized through cross-strand stacking and mismatch formation in 10 mM Na(+) solution (reported previously), to a dimeric hexad motif stabilized by extensive inter-subunit stacking of symmetry-related A.(G.G.G.G).A hexads in 150 mM Na(+) solution. Potential monovalent cation binding sites within the arrowhead and hexad motifs have been probed by a combination of Brownian dynamics and unconstrained molecular dynamics calculations. We could not identify stable monovalent cation-binding sites in the low salt arrowhead motif. By contrast, five electronegative pockets were identified in the moderate salt dimeric hexad motif. Three of these are involved in cation binding sites sandwiched between G.G.G. G tetrad planes and two others, are involved in water-mediated cation binding sites spanning the unoccupied grooves associated with the adjacent stacked A.(G.G.G.G).A hexads. Our demonstration of A.(G. G.G.G).A hexad formation opens opportunities for the design of adenine-rich G-quadruplex-interacting oligomers that could potentially target base edges of stacked G.G.G.G tetrads. Such an approach could complement current efforts to design groove-binding and intercalating ligands that target G-quadruplexes in attempts designed to block the activity of the enzyme telomerase.     

An intramolecular quadruplex of (GGA)(4) triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.     

Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2

A total of 26 polypeptides have been resolved by gel electrophoresis of purified phage PBSX, 3 of which belong to the head and the remainder to the tail. After mitomycin C treatment, synthesis of 11 additional proteins which are not found in the assembled phage particle was demonstrated, all but 4 being under the control of the phage repressor. Existence of a prehead and of a precursor of the main capsid protein (molecular weight, 35,000) suggested phage head maturation which is accompanied by cleavage of the precursor (molecular weight, 36,500). The role of induced proteins related and unrelated to PBSX is discussed. Finally, the estimated phage genome mass of 4 X 10(7) daltons exceeded by more than four times its head capacity, which could explain the defectiveness of the phage.     

Purification and characterization of epidermal G2- and G1-chalones]

Highly purified epidermal G1- and G2-chalones from rat skin inhibit the entering of epidermocytes to S and M phases of cell cycle respectively. Their biological activity is characterized by tissue-specificity and not by species-specificity. Both of them are tissue-specific glycoproteins as for their antigenic properties. Molecular weight of G1-chlone is 21 000, G2-chalone--34 000, isoelectric point (pH) 5.55 and 5.85 respectively. G2-chalone is the fastest as compared to G1-chalone in 5% acrylamide gel electrophoresis, pH 8.3. When injected in rabbits, G2-chalone produced monospecific antibodies which have no cross-reactivity with G1-chalone. The amino acid composition of both chalones and immunofluorescent localization of G2-chalone in epidermal tisues are given.     

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

Pollination in Geonoma macrostachys and Three Congeners,G. acaulis,G. gracilis,and G. interrupta

A continuous 15 month study of the floral ecology of four syntopic understorey palm species of Genoma was conducted in Amazonian Peru lowland rainforest. The spicate inflorescences of G. macrostachys, G. acaulis and G. gracilis are strictly protandrous and the plants are functionally dioecious. Data suggest that in G. macrostachys and G. acaulis pollination is based on a mimicry system, the pistillate flowers mimicking the staminate ones in colour, shape and scent. Pollen-collecting meliponine bees (Hymenoptera, Apidae, Meliponinae) and pollen-feeding syrphid flies (Diptera, Syrphidae) which visit inflorescences during both sexual stages are the pollinators of G. macrostachys. Geonoma acaulis is pollinated by small pollen-feeding weevils (Coleoptera, Curculionidae, Derelomini) that visit male and female spikes. Additionally, in G. macrostachys another pollinator type, viz. euglossine bees (Hymenoptera, Apidae, Euglossinae), which are attracted and rewarded by both types of flowers may account for long-distance pollination. The palm G. gracilis shows a very distinct pollination system. Although opportunistic insect visitors are attracted to the inflorescences of this species it seems to be mainly anemophilous because pollen becomes powdery during an thesis. The branched inflorescences of G. interrupta are also protandrous, but unlike the other species of Geonoma observed, staminate and pistillate anthesis of individual flowers are, for the most, overlapping. A broad spectrum of visitors is attracted (bees, wasps, flies, and beetles), which all may act as pollinators. Outcrossing is especially encouraged during the purely female phase at the end of the flowering cycle when there are no more staminate flowers in the inflorescence. Effects on the reproductive biology and population structure of different pollination systems and breeding system are discussed.     

G beta association and effector interaction selectivities of the divergent G gamma subunit G gamma(13).

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.