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At least 595 records · Page 33Linked to original sources
PubMed2006

TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes.

Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 microm, 25 nm, 1.5 microm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Animals↗
PubMed2006

A constitutive model of the human vocal fold cover for fundamental frequency regulation.

The elastic as well as time-dependent mechanical response of the vocal fold cover (epithelium and superficial layer of the lamina propria) under tension is one key variable in regulating the fundamental frequency. This study examines the hyperelastic and time-dependent tensile deformation behavior of a group of human vocal fold cover specimens (six male and five female). The primary goal is to formulate a constitutive model that could describe empirical trends in speaking fundamental frequency with reasonable confidence. The constitutive model for the tissue mechanical behavior consists of a hyperelastic equilibrium network in parallel with an inelastic, time-dependent network and is combined with the ideal string model for phonation. Results showed that hyperelastic and time-dependent parameters of the constitutive model can be related to observed age-related and gender-related differences in speaking fundamental frequency. The implications of these findings on fundamental frequency regulation are described. Limitations of the current constitutive model are discussed.

Adolescent↗
PubMed2002

Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes.

Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M5 muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr-->TyrPro was introduced into M1 and M5, and AsnThr-->TyrPro was introduced into M2, M3, and M4. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M2 receptor to 15- to 20-fold at M1, M3, and M4, to 40-fold at M5. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80% of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M5 subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.

3T3 Cells↗
PubMed2006

An essential role for constitutive endocytosis, but not activity, in the axonal targeting of the CB1 cannabinoid receptor.

In central neurons, the cell-surface distribution of cannabinoid receptor subtype-1 (CB(1)) is highly polarized toward axons and is associated with synaptic terminals, in which it is well-positioned to modulate neurotransmitter release. It has been suggested that high levels of constitutive activity mediate CB(1) receptor axonal targeting, leading to domain-specific endocytosis. We have investigated further the mechanisms that underlie CB(1) receptor axonal polarization in hippocampal neurons and found that constitutive activity is not an essential requirement for this process. We demonstrate that the cell-surface distribution of an N-terminally tagged, fluorescent CB(1) receptor fusion-protein is almost exclusively localized to the axon when expressed in cultured hippocampal neurons. Inhibition of endocytosis by cotransfection with a dominant-negative dynamin-1 (K44A) mutant traps both recombinant and endogenous CB(1) receptors at the somatodendritic cell surface. However, this effect could not be mimicked by inhibiting constitutive activity or receptor activation, either by expressing mutant receptors that lack these properties or by treatment with CB(1) receptor antagonists possessing inverse agonist activity. These data are consistent with a revised model in which domain-specific endocytosis regulates the functional polarization of CB(1) receptors, but this process is distinct from constitutive activity.

Animals↗
PubMed2000

Pharmacological properties of 5-Hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors.

We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.

Adenylyl Cyclases↗
PubMed2001

Agonism, inverse agonism, and neutral antagonism at the constitutively active human neurotensin receptor 2.

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.

Amino Acid Sequence↗
PubMed2004

Random mutagenesis of the human adenosine A2B receptor followed by growth selection in yeast. Identification of constitutively active and gain of function mutations.

To gain insight in spontaneous as well as agonist-induced activation of the human adenosine A2B receptor, we applied a random mutagenesis approach in yeast to create a large number of receptor mutants and selected mutants of interest with a robust screening assay based on growth. The amino acid sequence of 14 mutated receptors was determined. All these mutated receptors displayed constitutive activity. In particular, single-point mutations at T42A, V54L, and F84S and a triple-point mutation at N36S, T42A, and T66A resulted in high constitutive activity. In addition, a C-terminally truncated (after Lys269) mutant, Q214L I230N V240M V250M N254Y T257S K269stop, was highly constitutively active. The T42A, V54L, and F84S mutants showed a considerable decrease, 4.9- to 6.9-fold, in the EC50 value of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog. Combined mutation of I242T, K269R, V284A, and H302Q, as well as F84L together with S95G, resulted in an even greater potency of NECA of 10- and 18-fold, respectively. In fact, all constitutively active mutants had an increased potency for NECA. This suggests that the wild-type (wt) human A2B receptor itself is rather silent, which may explain the low affinity of agonists for this receptor. To verify the ability of the mutant receptors to activate mammalian second messenger systems, cAMP experiments were performed in CHO cells stably expressing the wt and T42A receptors. These experiments confirmed the increased sensitivity of T42A for NECA, because the EC50 values of T42A and the wt receptor were 0.15 +/- 0.04 and 1.3 +/- 0.4 microM, respectively.

Amino Acid Sequence↗
PubMed1976

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: constitutive mutants specifically derepressed for uidA expression.

All methyl-beta-D-galacturonide-positive mutants isolated from Escherichia coli K-12 carry constitutive mutations for beta-glucuronidase (UID) synthesis. Most of these mutants are specific for UID synthesis and are distributed in three classes according to the derepression level of UID. Each specific mutant carries a mutation(s) near uidA, the structural gene for UID, at min 30.5 of the E. coli K-12 linkage map. The expression of UID synthesis in F-merodiploid strains carrying these mutations permits discrimination between dominant and recessive constitutivity over the wild-type allele. The first kind of mutation (dominant) should affect the operator site uidO of the structural gene uidA; the second type of mutation (recessive) should affect a regulatory gene, uidR, operating through a negative control. The isolation of mutants bearing at this locus superrepressed mutations, which can revert to produce a constitutive phenotype, confirms the occurrence of such a regulatory gene. The partially derepressed uidR mutants of the first class are normally inducible and remain constitutive at low temperature; their UID has the same thermal sensitivity as in the wild-type strains. The occurrence of similar regulatory gene mutants has been recently described in the lactose system (Shineberg, 1974).

Cell-Free System↗
PubMed1976

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.

Aldehyde Oxidoreductases↗
PubMed1988

Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A.

The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM).

Alcaligenes↗
PubMed1992

Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene.

The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations.

Alleles↗
PubMed1966

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. 3. Isolation and properties of constitutive mutants.

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. III. Isolation and properties of constitutive mutants. J. Bacteriol. 91:1161-1167. 1966.-Mutants of Pseudomonas putida constitutive for the synthesis of l(+)-mandelate dehydrogenase were obtained after mandelate- or benzoylformate-limited growth in a chemostat. When grown in media noninducing for the wild type, the mutants are capable of coordinate, constitutive synthesis of the first five enzymes of the mandelate pathway. Later enzymes of the pathway that were examined are normally repressed. The constitutive mutants have two other noteworthy properties: they are superinducible by some compounds which induce the mandelate group enzymes in the wild type, or as a result of exhaustion of the carbon and energy source of the medium in which they are grown; and they exhibit a decreased specificity of induction, being inducible by a wide range of compounds devoid of inductive function for the wild type. These results, together with other evidence indicating that the five mandelate group enzymes comprise a regulatory unit, are discussed and evaluated in the context of the general problem of the regulation of complex dissimilatory pathways.

Carboxy-Lyases↗
PubMed1986

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.

Animals↗
PubMed1989

Identification of a novel constitutive enhancer element and an associated binding protein: implications for human papillomavirus type 11 enhancer regulation.

The human papillomavirus type 11 enhancer, when linked to the minimal simian virus 40 early promoter, has been dissected into two domains in monkey kidney CV-1 cells, one being constitutive (designated CEI) and the other inducible by trans-acting E2 proteins encoded by homologous and heterologous papillomaviruses (H. Hirochika, T.R. Broker, and L.T. Chow, J. Virol. 61:2599-2606, 1987; H. Hirochika, R. Hirochika, T.R. Broker, and L.T. Chow, Genes Dev. 2:54-67, 1988). We have demonstrated that the natural promoter regulated by this enhancer is located immediately upstream of the E6 open reading frame (the E6 promoter). We have mapped the cap site to nucleotide 99 by RNase protection. We further demonstrate a second constitutive enhancer element, CEII, which is required for transcription from the E6 promoter in the human cervical carcinoma cell lines C-33A and HeLa but not in CV-1 cells. By deletion mapping, we have localized this cell type-specific domain to 71 base pairs by using chloramphenicol acetyltransferase assays. Deletion of either CEI or CEII dramatically decreased the constitutive activity of the enhancer and the E6 promoter, whereas multimerization of either domain in the absence of the other could independently restore expression. Furthermore, when either of these elements was deleted, the full-length E2 protein of human papillomavirus type 11 abolished the remaining basal E6 promoter activity, demonstrating for the first time that the enhancer-activating E2 protein of human papillomaviruses can also function as a transcriptional repressor for the homologous E6 viral promoter. The presence of multiple copies of each element in tandem overcomes the repression by the E2 protein. The effects of CEII are at the level of transcription, without changing the cap site. By gel shift assay, we have shown that a protein present in nuclear extracts of C-33A and HeLa cervical carcinoma cells binds to the newly identified constitutive element II. This protein did not bind the simian virus 40 enhancer, nor did it bind to the enhancer region of many other papillomaviruses tested. UV cross-linking experiments revealed major 44-kilodalton and minor 34-kilodalton proteins that bound specifically to CEII. These two proteins are either related or bind to CEII with high cooperativity. We conclude that transcriptional activities directed by the enhancer and E6 promoter reflect an intricate balance among viral and cellular factors. We present a model on the regulation of the E6 promoter by host and viral transcription factors.

Animals↗
PubMed1992

Constitutive expression of an ISGF2/IRF1 transgene leads to interferon-independent activation of interferon-inducible genes and resistance to virus infection.

Interferon (IFN)-stimulated gene factor 2 (ISGF2) plays a role in transcription of the beta IFN (IFN-beta) gene and IFN-stimulated genes (ISGs) and may function as a central mediator of cytokine responses. Constitutive ISGF2 transgene expression resulted in substantial resistance to three RNA virus families. This phenotype was not a consequence of IFN production and may have arisen directly through ISG expression. ISGF2 acted generally as a positive transcription factor through binding sites from several genes, in the context of transient cotransfection. Constitutive transcription of the endogenous IFN-beta gene, and several genes that are normally induced by either IFN-alpha or IFN-gamma, or only by IFN-alpha, was elevated in cells that constitutively express an ISGF2 transgene. However, constitutive and virus-induced levels of IFN-beta mRNA were unaffected in such cell lines.

Antibodies, Viral↗
PubMed1998

Both the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus induce constitutive activation of the Raf-1/mitogen-activated protein kinase signal transduction pathway.

The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an attempt to understand how the virus causes Epo independence, we have been studying signal transduction pathways activated by Epo to determine if SFFV exerts its biological effects by constitutively activating any of these pathways in the absence of Epo. We previously demonstrated that Stat proteins, the downstream components of the Epo-induced Jak-Stat pathway, are constitutively activated in SFFV-infected cells. In this study, we demonstrate that SFFV also activates Raf-1, MEK and mitogen-activated protein (MAP) kinase, the downstream components of the Raf-1/MAP kinase pathway. This pathway was activated in cells infected with the polycythemia-inducing strain of SFFV, which induces both proliferation and differentiation of erythroid cells in the absence of Epo, as well as in cells infected with the anemia-inducing strain of the virus, which still require Epo for differentiation. Inhibition of Raf-1 by using antisense oligonucleotides led to a partial inhibition of the Epo-independent proliferation of SFFV-infected cells. Expression of the transcription factors c-Jun and JunB, but not c-Fos, was induced in SFFV-infected cells in the absence of Epo, suggesting that constitutive activation of the Raf-1/MAP kinase pathway by the virus may result in deregulation of AP-1 activity. We conclude from our studies that infection of erythroid cells with SFFV leads to the constitutive activation of signal transduction molecules in both the Jak-Stat and Raf-1/MAP kinase pathways and that both of these pathways must be activated to achieve maximum proliferation and differentiation of erythroid cells in the absence of Epo.

Anemia↗
PubMed2003

Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28.

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1 alpha, and the CX(3)C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca(2+). Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G(q/11)-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5'-O-(3-[(35)S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.

Cells, Cultured↗
PubMed1991

Constitutively expressed c-myb abrogates the requirement for insulinlike growth factor 1 in 3T3 fibroblasts.

The proto-oncogene c-myb, whose expression is usually limited to cells of the hematopoietic lineages, can be expressed in fibroblasts if placed under the control of a constitutive promoter, such as the simian virus SV40 early promoter. 3T3 cells carrying a constitutively expressed human c-myb were found to grow in 1% serum or in a serum-free, platelet-derived growth factor-supplemented medium, whereas the parent cell line, BALB/c 3T3, needed insulinlike growth factor 1 (IGF-1) in addition to platelet-derived growth factor for growth. myb-carrying cells, however, could not grow in platelet-poor plasma. In fibroblasts, therefore, a constitutively expressed c-myb can abrogate the requirement for platelet-poor plasma or IGF-1. When 3T3 cells constitutively expressed both c-myc and c-myb, they could grow in serum-free medium without added growth factors. The ability of c-myb to abrogate in fibroblasts the IGF-1 requirement seems to be due to its ability to induce overexpression of IGF-1, as indicated by an increase in steady-state levels of IGF-1 mRNA. These results have some important implications; for instance, they suggest a commonality of pathways for entry into S phase in different cell types and the possibility of a myb-like or myb-equivalent gene product of critical importance for entry of fibroblasts into S phase.

Animals↗