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1.
Chen L Xie L Dai Y Xiong X Fan W Zhang R 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2004,139(4):669-679
Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization. 相似文献
2.
G proteins are heterotrimeric GTPases that play a key role in signal transduction. The α subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gsα subunits are 92% identical to those found in the short form of human Gsα. Despite this high homology, the X. laevis Gsα clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc− membranes in contrast with human Gsα similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gsα. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gsα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human Gsα in activating adenylyl cyclase. 相似文献
3.
A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous
layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate
the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre
biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1
preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline
phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged
after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization. 相似文献
4.
Min Chen Jie Wang Kathryn E. Dickerson James Kelleher Tao Xie Divakar Gupta Edwin W. Lai Karel Pacak Oksana Gavrilova Lee S. Weinstein 《Cell metabolism》2009,9(6):548-555
In Albright hereditary osteodystrophy, a monogenic obesity disorder linked to heterozygous mutations of Gsα, the G protein that mediates receptor-stimulated cAMP generation, obesity develops only when the mutation is on the maternal allele. Likewise, mice with maternal (but not paternal) germline Gsα mutation develop obesity, insulin resistance, and diabetes. These parent-of-origin effects are due to Gsα imprinting, with preferential expression from the maternal allele in some tissues. As Gsα is ubiquitously expressed, the tissue involved in this metabolic imprinting effect is unknown. Using brain-specific Gsα knockout mice, we show that Gsα imprinting within the central nervous system underlies these effects and that Gsα is imprinted in the paraventricular nucleus of the hypothalamus. Maternal Gsα mutation impaired melanocortin stimulation of energy expenditure but did not affect melanocortin's effect on food intake, suggesting that melanocortins may regulate energy balance in the central nervous system through both Gsα-dependent and -independent pathways. 相似文献
5.
Anthony Yiu-Ho Woo Krzysztof Jozwiak Lawrence Toll Mary J. Tanga Joseph A. Kozocas Lucita Jimenez Ying Huang Ying Song Anita Plazinska Karolina Pajak Rajib K. Paul Michel Bernier Irving W. Wainer Rui-Ping Xiao 《The Journal of biological chemistry》2014,289(28):19351-19363
Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4′-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R′)-4′-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism. 相似文献
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Takako Naganuma Wataru Hoshino Yukihiro Shikanai Rie Sato Kaiyue Liu Saho Sato Koji Muramoto Makoto Osada Kyosuke Yoshimi Tomohisa Ogawa 《PloS one》2014,9(11)
Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO3 crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation. 相似文献
8.
Richard Carr III Yang Du Julie Quoyer Reynold A. Panettieri Jr. Jay M. Janz Michel Bouvier Brian K. Kobilka Jeffrey L. Benovic 《The Journal of biological chemistry》2014,289(52):35668-35684
The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β2AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β2AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β2AR and provides valuable tools for the study of β2AR function. 相似文献
9.
Hiroshi Itoh Toshiaki Katada Michio Ui Hiroshi Kawasaki Koichi Suzuki Yoshito Kaziro 《FEBS letters》1988,230(1-2)
We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2α gene and rat Gi2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1α and Goα cDNAs, respectively. 相似文献
10.
Yaohua Shi Xing Zheng Xin Zhan Aimin Wang Zhifeng Gu 《Marine biotechnology (New York, N.Y.)》2016,18(3):336-348
Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E?≤??10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster. 相似文献
11.
Partha Sinha Piia Aarnisalo Rhiannon Chubb Ingrid J. Poulton Jun Guo Gregory Nachtrab Takaharu Kimura Srilatha Swami Hamid Saeed Min Chen Lee S. Weinstein Ernestina Schipani Natalie A. Sims Henry M. Kronenberg Joy Y. Wu 《The Journal of biological chemistry》2016,291(4):1631-1642
Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-GsαOsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-GsαOsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-GsαOsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation. 相似文献
12.
Hoa T.N. Phan Joseph Loomis Saji Abraham Qing He Murat Bastepe Alan V. Smrcka 《The Journal of biological chemistry》2022,298(8)
Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein–coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology. 相似文献
13.
Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The α subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the Gα family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only 10% of total Gα protein expressed is active; the rest accumulates in inclusion bodies. Although Giα has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold Gα from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of Gα subunits (human Giα(1), human Gsα(short), human G11α and human Gtα(cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble Giα expressed from E. coli showed that although the refolded Gα subunits were soluble and retained partial α-helicity characteristic of the native, folded Gα subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded Giα protein has a native-like secondary structure, but is predominately in a molten globular state. 相似文献
14.
Alkaline phosphatases are ubiquitous enzymes found in most species including the pearl oyster, Pinctada fucata, where it is presumably involved in nacreous biomineralization processes. In the present study, we have purified alkaline
phosphatases from the pearl oyster and modified the tryptophan residues using N-bromosuccinimide (NBS). We show that the resulting
inactivation of purified alkaline phosphatase by NBS is dependent on modification of only one of five tryptophan residues
in the enzyme. Substrate protection experiments showed that the tryptophan residue was not located at the substrate-binding
site but was involved in the catalytic activity.
Published in Russian in Biokhimiya, 2008, Vol. 73, No. 1, pp. 107–112. 相似文献
15.
Eyvind J. Paulssen Ruth H. Paulssen Kaare M. Gautvik Jan O. Gordeladze 《Cellular signalling》1992,4(6)
We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (Gsα and Gi−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated Gsα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered Gsα levels by more than 50% at all time-points. Gi−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane Gs protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways. 相似文献
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The heterotrimeric G proteins are known to have a variety of downstream effectors, but Gs was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated Gs 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 Gs-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 Gαs subfamily regulates activity of Rho GTPases extends our understanding of Gαs activity and establishes RhoGEF coupling as a universal Gα function.The canonical G protein pathway consists of a cell surface receptor, a heterotrimeric G protein, and an effector protein that controls signaling within the cells. This fundamental paradigm, familiar to every biologist, is rooted in discoveries by the laboratories of Sutherland, Rodbell, and Gilman, which in the 1970s and 1980s dissected biochemical mechanisms of adenylyl cyclase activation by hormones. Their breakthrough came after experiments showing that the G protein Gs is essential to transfer agonist stimulation from the receptor to adenylyl cyclase (1). This G protein consists of the ∼42-kDa α subunit, which binds and hydrolyzes GTP, and the permanently associated dimer of 35-kDa β and ∼10-kDa γ subunits (Gβγ). Their findings helped establish a canonical model in which the agonist-bound receptor causes the G protein to release GDP, and the heterotrimer dissociates into Gα-GTP and free Gβγ; in this state, the G protein can activate its effector (i.e. Gαs will activate adenylyl cyclase until GTP is hydrolyzed). Although the rod photoreceptor G protein, transducin, was discovered by that time (2), the ubiquitously expressed Gs can be considered the founding member of the G protein family.The subsequent cloning and identification of the other three families (Gi, Gq, and G12) completed the rough map of G protein–mediated transduction. These initial studies suggested that the α subunits were responsible for activation of one type of effector (e.g. Gαs for adenylyl cyclase and cAMP; Gαq for phospholipase C, phosphoinositides, and Ca2+; and Gαi for ion channels and inhibition of adenylyl cyclase), whereas the free Gβγ complexes interact with a remarkably large number of binding partners, including some effector enzymes and ion channels (3). Later, Gα12 and Gα13 were found to regulate a distinct type of effectors, the RhoGEFs (4, 5). These multidomain proteins contain pleckstrin homology (PH) domains, which facilitate their membrane localization, and Dbl homology (DH) domains, which catalyze GDP-for-GTP exchange (guanine nucleotide exchange factor; GEF) in the Rho family of small (∼20-kDa) G proteins. At the time, the G12-RhoGEF pathway seemed odd as it contained two G proteins: the receptor-activated “large” G12 class protein and the “small” Rho G protein, which is activated by RhoGEF. However, it was then discovered that Gαq could activate a RhoGEF called Trio (6), and that Gβγ complexes activate other RhoGEFs, indicating that this pathway, if unusual, is at least popular. Gαs, however, mostly appeared to be faithful to its originally determined role—to stimulate adenylyl cyclase(s)—possibly contributing to the enduring perception that regulation of a second messenger–generating enzyme is the “real” function of a heterotrimeric G protein.In the current issue of JBC, Castillo-Kauil et al. (7) force a reexamination of the existing canon, presenting data that show Gαs can also interact with a specific RhoGEF, in this case PDZ-RhoGEF (PRG). The authors made this discovery as part of an examination of the regulation of cell shape by the Rho family. They began by expressing a series of short constructs of three RhoGEF proteins, p115RhoGEF, PRG, and LARG, all of which activated RhoA as expected, promoting cell contraction. However, they noticed that the DH/PH domain of PRG also activated Cdc42 and induced filopodia-like cell protrusions. To investigate which G protein is responsible for activation of this Cdc42-mediated pathway, they overexpressed constitutively active mutants of different Gα subunits. These mutants are stabilized in the active GTP-bound state due to substitution of the glutamine residue crucial for GTP hydrolysis. Surprisingly, the PRG-Cdc42 pathway was stimulated by GαsQ227L, the one Gα subtype not known for interaction with RhoGEFs. Furthermore, they showed that binding of PRG to Cdc42 was promoted only by Gs-coupled receptors, and not by Gq- or Gi-coupled GPCRs. The authors then investigated the PRG site responsible for the interaction with Gαs, narrowing it down to the isolated PRG DH and PH domains and their linker region. A construct encompassing these domains was able to inhibit (i) GPCR-mediated activation of Cdc42, (ii) the GαsQ227L-promoted interaction of PRG with Cdc42, and (iii) some protein phosphorylation events downstream of the canonical cAMP pathway. Taken together, their work identifies PRG as a novel effector for Gs; the Gαs-PRG interaction mediates activation of Rho family protein Cdc42, leading to cytoskeletal remodeling.The unexpected results of Castillo-Kauil et al. open up new opportunities to explore this mechanism at different levels of biology. The experiments described in the paper were performed in vitro using cultured cells, imaging, and pulldown of protein complexes containing the overexpressed Gαs Q227L mutant. Considering the multitude of Gs-coupled receptors and RhoGEFs in the body (8, 9), it will be important to understand the physiological context where the new Gs-mediated pathway plays a significant role. This will require experimentation in vivo and possibly reevaluation of the phenotypes associated with known pathogenic mutations in Gαs (GNAS) and other relevant genes. At the molecular level, it would be important to delineate the biochemical mechanisms of Gαs interaction with PRG. For example, at what stage of the GTP/GDP cycle does Gαs bind to PRG: in the GTP-bound state, which also activates adenylate cyclase, or in the transition state (i.e. just before the terminal phosphate of GTP is removed)? Indeed, there is precedent for proteins that bind preferentially with the transition state—specifically RGS proteins, which accelerate the GTPase reaction. Another possibility is that, by analogy with p115RhoGEF, which stimulates GTPase activity of Gα12 and Gα13, PRG (and other RhoGEFs with similar DH-PH sequences) can influence interaction of Gαs with nucleotides, Gβγ, and other partners.Since defining the receptor, G protein, and effector as the three essential members of the G protein pathway, researchers have discovered many additional proteins that regulate the amplitude and duration of the stimulus and/or participate in cross-talk with other signaling circuits. These “new” proteins include arrestins, receptor kinases, nonreceptor exchange factors, GTPase-activating proteins, special chaperones, etc. Thus, in a way, discovering a novel binding partner for a signaling molecule is not as surprising as it would have been 20 years ago. However, the new partner identified by Castillo-Kauil et al. makes the result of extra significance; until now, we knew that three of four G protein subfamilies could regulate Rho GTPases by activating RhoGEFs: G12 and Gq via their α subunits and Gi via the Gβγ subunits (10). The demonstration that the Gs subfamily is no exception shows that activation of RhoGEFs by heterotrimeric G proteins may be a truly universal mechanism (Fig. 1). The significance of this insight is that the multitude of biological processes regulated by Rho-GTPase networks can potentially respond to the entire repertoire of GPCR-mediated stimuli.Open in a separate windowFigure 1.Activation of the Rho family by heterotrimeric G proteins. The Rho family of small GTPases is activated by RhoGEF proteins, some of which can be stimulated by heterotrimeric G proteins. Of four families of heterotrimeric G proteins, three (G12, Gq, and Gi, shown in shades of gray) were known to activate certain RhoGEFs. The new results (highlighted in orange) (7) show that Gs, the G protein known to stimulate production of cAMP, can also stimulate a particular RhoGEF; this suggests that the Rho GTPases can potentially be stimulated by the multitude of signals from the entire class of GPCRs, including those coupled to Gs. IP3, inositol 1,4,5-trisphosphate.Funding and additional information—This work was supported in part by National Institutes of Health Grant R56DK119262 (to V. Z. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.Abbreviations—The abbreviations used are:
- PH
- pleckstrin homology
- DH
- Dbl homology
- GEF
- guanine nucleotide exchange factor
- PRG
- PDZ-RhoGEF
- GPCR
- G protein–coupled receptor.
19.
Kishimura H Ojima T Hayashi K Nishita K 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2000,126(4):579-586
Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A2 (PLA2), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA2 protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA2 protein, suggesting the presence of multiple forms in the starfish PLA2. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA2. In phylogenetic tree, the starfish PLA2 should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA2, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA2 from porcine pancreas. 相似文献
20.
Mario Pagano J. Dedrick Jordan Susana R. Neves Tracy Nguyen Ravi Iyengar 《Cellular signalling》2008,20(6):1190-1197
The Gs and Gi pathways interact to control the levels of intracellular cAMP. Although coincident signaling through Gs and Gi-coupled receptors can attenuate Gs-stimulated cAMP levels, it is not known if prior activation of the Gi pathway can affect signaling by Gs-coupled receptors. We have found that activated Gαo/i interact with RGS20, a GTPase activating protein for members of the Gαο/i family. Interaction between Gαo/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gαo and Gαi2 but not by Gαq, Gαi1 or Gαi3. The Gαo/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gαo stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to Go/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that Go/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that Go/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the Gs and Gi pathways controls cellular cAMP levels, thus allowing for signal integration. 相似文献