共查询到20条相似文献,搜索用时 91 毫秒
1.
《Life sciences》1995,57(18):1675-1681
Various heterotrimeric GTP-binding proteins (G proteins) are possible to have important functions in hematopoietic cells. However, there has been no information regarding their expression in magakaryoblasts and/or megakaryocytes. In the present study, protein contents of seven G protein α subunits (Gs α, Gi2 α, Gi3 α, Gz α, G11 α, Gq α and G12 α) and β subunit in a human megakaryoblastic leukemia cell line, MEG-01, were analyzed by immunoblotting. Immature MEG-01 cells expressed the α subunits of Gs, Gi2, Gi3, Gz, G11 and G12 at protein molecule level. During the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced differentiation process, the contents of Gi2 α and Gi3 α increased, whereas the protein levels of Gz α, Gs α, Gil a and G12 α were observed to hardly change, β Subunit was also observed to be present in immature MEG-01 cells and to increase continuously throughout the differentiation process. For the expression of Gi2 α and β subunits, chronic TPA-treatment was required although Rac2, a low Mr GTP-binding protein, was expressed abundantly by only 30 min-TPA-treatment followed by 3 day-culture. 相似文献
2.
Membrane and cytosolic fractions prepared from ventricular myocardium of young (21-day-old) hypo- or hyperthyroid rats and adult (84-day-old) previously hypo- or hyperthyroid rats were analyzed by immunoblotting with specific anti-G-protein antibodies for the relative content of Gs alpha, Gi alpha/Go alpha, Gq alpha/G11 alpha, and G beta. All tested G protein subunits were present not only in myocardial membranes but were at least partially distributed in the cytosol, except for Go alpha2, and G11 alpha. Cytosolic forms of the individual G proteins represented about 5-60% of total cellular amounts of these proteins. The long (Gs alpha-L) isoform of Gs alpha prevailed over the short (Gs alpha-S) isoform in both crude myocardial membranes and cytosol. The Gs alpha-L/Gs alpha-S ratio in membranes as well as in cytosol increased during maturation due to a substantial increase in Gs alpha-L. Interestingly, whereas the amount of membrane-bound Gi alpha/Go alpha and Gq alpha/G11 alpha proteins tend to lower during postnatal development, cytosolic forms of these G proteins mostly rise. Neonatal hypothyroidism reduced the amount of myocardial Gs alpha and increased that of Gi alpha/Go alpha proteins. By contrast, neonatal hyperthyroidism increased expression of Gs alpha and decreased that of Gi alpha and G11 alpha in young myocardium. Changes in G protein content induced by neonatal hypo- and hyperthyroidism in young rat myocardium were restored in adulthood. Alterations in the membrane-cytosol balance of G protein subunits associated with maturation or induced by altered thyroid status indicate physiological importance of cytosolic forms of these proteins in the rat myocardium. 相似文献
3.
Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles. 总被引:3,自引:0,他引:3
The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4. 相似文献
4.
Decreased levels of guanyl nucleotide-binding regulatory protein alpha-subunits in Y1 adrenocortical tumor cell mutants resistant to forskolin. 总被引:1,自引:0,他引:1
Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane. 相似文献
5.
Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
G Kalinec A J Nazarali S Hermouet N Xu J S Gutkind 《Molecular and cellular biology》1992,12(10):4687-4693
The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene. 相似文献
6.
Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli 总被引:13,自引:0,他引:13
M E Linder D A Ewald R J Miller A G Gilman 《The Journal of biological chemistry》1990,265(14):8243-8251
Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity. 相似文献
7.
R Morishita Y Fukada K Kokame T Yoshizawa K Masuda M Niwa K Kato T Asano 《European journal of biochemistry》1992,210(3):1061-1069
Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit. 相似文献
8.
The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S. 相似文献
9.
Changes in the relative abundance of the G protein alpha subunits were observed during early mouse development Gs alpha was almost exclusively present as a large form (Gs-1) in prenatal brain. Postnatally with a substantial increase in Gpp[NH]p stimulated adenylyl cyclase activity, the small form (Gs.s) increased in amount while Gs-1 decreased. These results suggest that the Gs-s may be the more effective cyclase activator and that changes in alternative splicing are developmentally regulated. Gi1 and Go appeared before birth whereas Gi2 developed postnatally. Opiate stimulation of GTPase and inhibition of adenylyl cyclase were fully expressed prenatally. 相似文献
10.
Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system 总被引:4,自引:0,他引:4
W F Simonds R M Collins A M Spiegel M R Brann 《Biochemical and biophysical research communications》1989,164(1):46-53
Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits. 相似文献
11.
Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics 总被引:4,自引:0,他引:4
The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel. 相似文献
12.
We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists. 相似文献
13.
Proteins containing G-protein regulatory (GPR) motifs represent a novel family of guanine nucleotide dissociation inhibitors (GDIs) for G(alpha) subunits from the Gi family. They selectively interact with the GDP-bound conformation of Gi(alpha) and transducin-alpha (Gt(alpha)), but not with Gs(alpha). A series of chimeric proteins between Gi(alpha)(1) and Gs(alpha) has been constructed to investigate GPR-contact sites on G(alpha) subunits and the mechanism of GPR-protein GDI activity. Analysis of the interaction of two GPR-proteins-AGS3GPR and Pcp2-with the chimeric G(alpha) subunits demonstrated that the GPR-Gi(alpha)(1) interface involves the Gi(alpha)(1) switch regions and Gi(alpha)(1)-144-151, a site within the helical domain. Residues within Gi(alpha)(1)-144-151 form conformation-sensitive contacts with switch III, and may directly interact with a GPR-protein or form a GPR-binding surface jointly with switch III. The helical domain site is critical to the ability of GPR-proteins to act as GDIs. Our data suggest that a mechanism of the GDI activity of GPR-proteins is different from that of GDIs for monomeric GTPases and from the GDI-like activity of G(betagamma) subunits. The GPR-proteins are likely to block a GDP-escape route on G(alpha) subunits. 相似文献
14.
G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization 总被引:2,自引:0,他引:2
The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues. 相似文献
15.
G-protein mRNA levels during adipocyte differentiation 总被引:1,自引:0,他引:1
D C Watkins P J Rapiejko M Ros H Y Wang C C Malbon 《Biochemical and biophysical research communications》1989,165(3):929-934
G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells. 相似文献
16.
We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli. 相似文献
17.
The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2. 总被引:3,自引:0,他引:3
Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2. 相似文献
18.
GTP binding proteins: a key role in cellular communication 总被引:1,自引:0,他引:1
One of the major steps in the understanding of the hormonal and sensory transduction mechanisms in eukaryotic cells has been the discovery of a family of GTP binding proteins which couple receptors to specific cellular effectors. The absolute requirement of GTP for hormonal stimulation of adenylate cyclase was the initial observation which led to the purification of the protein involved: Gs. Gs couples stimulatory receptors to adenylate cyclase. It is a heterotrimer composed of an alpha chain (45 or 52 kDa), a beta chain (35-36 kDa) and a gamma chain (8 kDa). Several other G proteins of known functions have been purified: Gi, which couples inhibitory receptors to adenylate cyclase, and transducin which couples photoexcited rhodopsin to cyclic GMP phosphodiesterase. Some G proteins of uncertain function have also been purified: Go, a G protein mainly localized in nervous tissues and Gp, a G protein isolated from placenta and platelets. All these G proteins have a common design. Like Gs they all consist of 3 chains: alpha, beta and gamma. The beta chains are nearly identical, whereas the gamma chains are more variable. The alpha chains are different, but share common domains (especially at the level of the GTP binding site). These domains of homologies are also similar to those of other GTP binding proteins, such as the product of the ras gene (p21) and the initiation or elongation factors. alpha Chains are also ADP ribosylated by bacterial toxins. Gs and transducin are targets for cholera toxin, whereas Gi, Go and transducin are targets for pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor. 相似文献
20.
Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells 总被引:1,自引:0,他引:1
Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems. 相似文献