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PubMed2003

The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis.

US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.

Amino Acid Sequence↗

PubMed2000

Level of interleukin-8 expression by metastatic human melanoma cells directly correlates with constitutive NF-kappaB activity.

The purpose of this study was to determine whether constitutive NF-kappaB activity regulates the expression level of interleukin-8 (IL-8) in metastatic human melanoma cells. Cultures of metastatic human A375 melanoma cells expressed higher levels of IL-8 mRNA and protein than nonmetastatic A375 human melanoma cells. No discernible differences in IL-8 half-life were found between metastatic and nonmetastatic cells, but cells that overexpressed IL-8 had a higher transcription rate and increased IL-8 promoter activity. Analysis of the IL-8 promoter using deletion mutants revealed that the region within -133 was essential for constitutive IL-8 promoter activity and that mutation of NF-kappaB binding sites eliminated the constitutive IL-8 promoter activity. The activation of constitutive IL-8 transcription directly correlated with the level of constitutive NF-kappaB activity. Transfection of melanoma cells with a dominant-negative mutant IkappaBalpha expression vector (pLXSN-IkappaBalphaM) significantly decreased the level of constitutive NF-kappaB activity and expression of IL-8, demonstrating that constitutive NF-kappaB/relA activities contribute to overexpression of IL-8 in highly metastatic human melanoma cells.

Base Sequence↗

PubMed1989

Spatial segregation of the regulated and constitutive secretory pathways.

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.

Adrenocorticotropic Hormone↗

PubMed2004

Association between iris constitution and apolipoprotein e gene polymorphism in hypertensives.

OBJECTIVE: Iridology is a complementary and alternative medicine (CAM) that involves the diagnosis of medical conditions by noting irregularities of the pigmentation in the iris. Iris constitution has a strong familial aggregation and heredity is implicated. Apolipoprotein E (apoE) gene polymorphism is one of the most well-studied genetic markers for vascular diseases, including hypertension. In this study, we investigated the relationship between iris constitution and apoE polymorphism in hypertensives. DESIGN AND SUBJECTS: We classified 87 hypertensives and 79 controls according to iris constitution and determined the apoE genotype of each individual. RESULTS: A significantly higher percentage of individuals with neurogenic constitutions was found in the hypertensive group when compared with the control group (chi(2) = 40.244, p < 0.001). In addition, a neurogenic constitution increased the relative risk for hypertension for subjects with an apo epsilon2 or an epsilon4 allele (chi(2) = 4.086, p = 0.049, odds ratio = 2.633, confidence interval = 1.004-6.905). CONCLUSIONS: Our results imply that a neurogenic iris constitution enhances the relative risk for hypertension in subjects with the apo epsilon2 or epsilon4 allele. Furthermore, we attempted to evaluate the efficacy of iris constitutional medicine and to find an association with hypertension.

Adult↗

PubMed1983

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization.

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.In growth chamber studies, the fully expanded first trifoliolate leaf from NO(3) (-)-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO(3) (-) in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO(3) (-)-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO(3) (-)-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO(3) (-), and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO((x))] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO(3) (-)-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO((x)) evolution and occurs only in physiologically young leaves.

Journal Article↗

PubMed1983

Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance.

Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO(3) (-)-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO(3) (-) in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.Urea-grown LNR-2 seedlings lack both inducible and constitutive NR activity, and were resistant to four days of treatment with 0.5 mm ClO(3) (-). Urea-grown wild-type seedlings, having only constitutive NR activity, developed ClO(3) (-) toxicity symptoms and suffered decreases in unifoliolate leaf NR activity and chlorophyll concentration. This suggests that (a) the reduction of ClO(3) (-) to ClO(2) (-) by NR is the major cause of ClO(3) (-) toxicity in soybeans and (b) the constitutive NR is active in situ.Segregation of the F(2) of reciprocal crosses between the wild type and the mutants indicated that absence of constitutive NR activity was controlled by a single recessive nuclear gene. Evolution of NO((x)) gas was also absent in these mutants, and this was found to be inherited jointly with constitutive NR activity: in 346 segregants, no recombinants were found. Allelism tests between LNR-2 and LNR-3, and LNR-2 and LNR-4, indicated that the constitutive NR mutation was at the same locus in each mutant.

Journal Article↗

PubMed1992

Pentoxifylline inhibits certain constitutive and tumor necrosis factor-alpha-induced activities of human normal dermal fibroblasts.

Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.

2',5'-Oligoadenylate Synthetase↗

PubMed2006

The difference between the constitutive and facultative skin color does not reflect skin phototype in Asian skin.

BACKGROUND/PURPOSE: The assessment of the sensitivity of human skin to ultraviolet (UV) radiation is important in the area of phototherapy, photodermatoses, photo-aging, photo-carcinogenesis, and photo-protection. Some reports have shown that quantitatively measured skin color is a good indicator for predicting UV sensitivity to human skin in Caucasians. In this study, our aim was to define the correlation between skin color and the skin phototype assessed by the Fitzpatrick method in Asian brown skin. METHODS: A total of 180 medical students with similar life styles were included in this study. Their skin phototype was classified according to the system introduced by Fitzpatrick. Then, using a Minolta Spectrophotometer CM-2002, their skin color was determined on the buttocks and forehead. The buttock color was taken as the constitutive skin color, and the forehead color as the facultative skin color. Using these measured values, we compared the skin color with the skin phototype to find their correlation. Also, we investigated whether the difference between the constitutive and facultative skin colors of each individual had a relationship with his or her skin phototype. RESULTS: The constitutive skin color became darker with increasing skin phototype, and this change was statistically significant. As for the facultative skin color, it also became darker with increasing skin phototype, but was less well correlated with the skin phototype than the constitutive skin color. However, the difference between the constitutive and facultative skin colors did not show consistent results in predicting the skin phototype. CONCLUSION: In this study, we found that the constitutive skin color can be a good indicator of the skin phototype. However, the difference between the constitutive and facultative skin colors of each individual does not give any meaningful information for the assessment of his or her skin phototype in Asian skin.

Adult↗

PubMed1997

Quantitative analysis of constitutive heterochromatin in couples with fetal wastage.

PROBLEM: Heteromorphism of constitutive heterochromatin is a stable evolutionary feature that is thought to cause no phenotypic alterations. Nevertheless, the role of constitutive heterochromatin is still unknown. The instability of constitutive heterochromatin was generally restricted to T-lymphocytes and was associated with variable immunodeficiency. The heterochromatin regions of chromosomes 1, 9, 16, and Y have been postulated to play a role in the immune response and during early embryo development. METHOD OF STUDY: To investigate a possible influence of constitutive heterochromatin in human reproductive ability, quantitative analysis of constitutive heterochromatin in human chromosomes 1, 9, 16 and Y was done. Thirty couples were divided into two groups, owing to the clinical heterogeneity of their reproductive disorders. The first group included couples with two or more spontaneous abortions as the only pregnancy outcomes, and the second group included couples with a stillborn child with or without malformations. In the control group were couples with one or more healthy children without a history of fetal wastage. All of the persons in this study had normal karyotypes. The amount of constitutive heterochromatin was expressed by relative value using the simple transformation [q/(p + q)]. This value, obtained on GTG-banded metaphase chromosomes, represented an indirect measure of heterochromatin content. The Y/F index was used to express the relative amount of heterochromatin in chromosome Y. RESULTS: There was a significant increase in the heterochromatin content of the chromosomes 16 homologue pair in males and females with a stillborn or a stillborn malformed child (P < 0.01) and an increase in total heterochromatin cell content compared to controls (P = 0.005). The same couples had significantly increased mean maximal heterochromatin content in the potential zygotes (P < 0.02). The couples who experienced spontaneous abortions only had a minimal total heterochromatin content in the potential zygotes (P < 0.05). The Y/F index was significantly lower in the males in both groups compared to controls (P1 < 0.02; P2 < 0.02). CONCLUSION: The quantitative analysis of constitutive heterochromatin could be valuable in predicting pregnancy outcome.

Abortion, Spontaneous↗

PubMed2005

Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels 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 that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel activity through activation of PKC. This study indicates that constitutive cation channel activity in ear artery myocytes is mediated by DAG which is generated by PC-PLD via phosphatidic acid which represents a novel activation pathway of cation channels in vascular myocytes.

Animals↗

PubMed2006

ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor.

The human adenosine A(2B) receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A(2B) receptors were identified from a random mutation bank. In the current study, three known A(2B) receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A(2B) receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A(2B) receptor because they were able to fully reverse the basal activity of four low-level constitutively active A(2B) receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A(2B) receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 +/- 0.03, 0.35 +/- 0.03, and 0.31 +/- 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A(2B) receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.

Adenosine A2 Receptor Agonists↗

PubMed2005

Carboxyl tail cysteine mutants of the thyrotropin-releasing hormone receptor type 1 exhibit constitutive signaling: role of palmitoylation.

We studied the role of carboxyl tail cysteine residues and their palmitoylation in constitutive signaling by the thyrotropin-releasing hormone (TRH) receptor type 1 (TRH-R1) in transfected mammalian cells and in Xenopus laevis oocytes. To study palmitoylation, we inserted a factor Xa cleavage site within the third extracellular loop of TRH-R1, added a carboxyl-terminal C9 immunotag and expressed the mutant receptor in Chinese hamster ovary cells. We identified TRH-R1-specific palmitoylation in the transmembrane helix-7/carboxyl-tail receptor fragment mainly at Cys-335 and Cys-337. In contrast to a mutant truncated at Cys-335 that was reported previously to be constitutively active, a receptor truncated at Lys-338 (K338Stop), which preserves Cys-335 and Cys-337, and C337Stop and N336Stop, which preserve Cys-335, did not exhibit increased constitutive signaling. TRH-R1 mutants substituted singly by Gly or Ser at Cys-335 or Cys-337 did not exhibit constitutive signaling. By contrast, substitution of both cysteines (C335G/C337G or C335S/C337S) yielded TRH-R1 mutants that exhibited marked constitutive signaling in mammalian cells. In the oocyte, constitutive signaling by C335G/C337G resulted in homologous (of C335G/C337G) and heterologous (of M1 muscarinic receptor) desensitization. Because both Cys-335 and Cys-337 have to be substituted or deleted for constitutive signaling, we propose that a single palmitoylation site in the proximal carboxyl tail is sufficient to constrain TRH-R1 in an inactive conformation.

Animals↗

PubMed2005

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor.

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.

Animals↗

PubMed2001

Histamine H(1)-receptor activation of nuclear factor-kappa B: roles for G beta gamma- and G alpha(q/11)-subunits in constitutive and agonist-mediated signaling.

Nuclear factor kappa B (NF-kappa B) is an important transcription factor in inflammation that has obtained a great interest as a drug target for the treatment of various allergic conditions. In this study, we show that the histamine H(1) receptor, which is also an important player in allergic and inflammatory conditions, activates NF-kappa B in both a constitutive and agonist-dependent manner. Moreover, the observed constitutive NF-kappa B activation is inhibited by various H(1)-receptor antagonists, suggesting that inverse agonism may account, at least in part, for their ascribed antiallergic properties. Investigation of the H(1) receptor-mediated NF-kappa B activation in transfected COS-7 cells indicates that the level of the observed constitutive activity of the H(1) receptor can be modulated by the expression levels of either G alpha-proteins or G beta gamma-heterodimers. Members of the G alpha(q/11)-family of G alpha-proteins are most effective in increasing H(1) constitutive activity. Also, coexpression of G beta(2) in combination with either G gamma(1) or G gamma(2) results in an increased constitutive activity of the H(1) receptor, whereas scavenging of G beta gamma-subunits by coexpression of G alpha(t) completely neutralizes the constitutive, but not the agonist-induced, NF-kappa B activity. Our data suggest that both G alpha(q/11)- and G beta gamma-subunits play a role in the agonist-induced, H(1) receptor-mediated NF-kappa B activation, but that constitutive NF-kappa B activation by the H(1) receptor is primarily mediated through G beta gamma-subunits.

Animals↗

PubMed2002

Murine cytomegalovirus (CMV) M33 and human CMV US28 receptors exhibit similar constitutive signaling activities.

Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-kappaB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-kappaB activation. Interestingly, UL33-the HCMV-encoded structural homologue of M33-was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-kappaB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.

Animals↗

PubMed2001

A two-locus model for experience-conditioned direction of paw usage in the mouse is suggested by dominant and recessive constitutive paw usage behaviours.

Left-right direction of paw usage in the mouse depends on the genotype and the directional nature of the test. There are two phenotypic classes; in some strains, direction of paw usage is learned or conditioned by the direction of the initial test chamber and the experience of reaching and, in other strains, paw usage is a constitutive behaviour not affected by previous experience. We report the evidence for locus heterogeneity in the cause of constitutive versus experience-conditioned paw usage from a phenotypic analysis of F1 hybrid generations from the experience-conditioned C57BL/6J, C3H/HeHa, and SWV strains and the constitutive CDS/Lay and DBA/2J strains. The F1 hybrids between strains of different phenotypic classes provide evidence of locus heterogeneity. Constitutive paw usage in CDS/Lay is phenotypically dominant to experience-conditioned behaviour in both C57BL/6J and SWV. However, constitutive paw usage in DBA/2J is phenotypically recessive to experience-conditioned behaviour in C57BL/6J and dominant to experience-conditioned behaviour in SWV. Among the experience-conditioned strains, C57BL/6J is highly lateralized but SWV is only weakly lateralized. Our data suggest a model in which C57BL/6J may have a "strong" allele that identifies a functional difference between the constitutive paw usage of CDS/Lay and DBA/2J. DBA/2J may have a loss-of-function mutation at the same locus that is recessive to the strong C57BL/6J allele. SWV may have a "weak" allele and the (SWV x D2)F1 compound heterozygote may be below a threshold for detectability of experience-conditioned behaviour, making the constitutive behaviour of DBA/2J appear to be dominant to the experience-conditioned behaviour of SWV. CDS/Lay may have a dominant allele at a second locus that suppresses experience-conditioned behaviour in all F1 hybrids.

Animals↗

PubMed2006

Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-kappaB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-kappaB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-kappaB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-kappaB. Activation of TG2 led to activation of NF-kappaB; conversely, inhibition of TG2 activity inhibited activation of NF-kappaB. Similarly, ectopic expression of TG2 caused activation of NF-kappaB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-kappaB. Our results further indicated that constitutive NF-kappaB reporter activity in pancreatic cancer cells is not affected by dominant-negative I kappaB alpha. Additionally, coimmunoprecipitation and confocal microscopy showed that I kappaB alpha is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-kappaB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-kappaB in tumor cells via a novel pathway that is most likely independent of I kappaB alpha kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-kappaB activation and rendering cancer cells sensitive to anticancer therapies.

Breast Neoplasms↗

PubMed1995

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin.

To evaluate the role of protein aggregation and calcium in the sorting of insulin for regulated vs. constitutive release from the intact pancreas, we targeted the expression of a monomeric mutant form of human (pro)insulin (B9/B27) to the pancreatic beta-cells of transgenic mice. This mutant insulin does not form dimers or hexamers, but can aggregate at high concentration in the presence of calcium. A homozygous line (171) was produced that expressed 55% of the total (pro)insulin message in their beta-cells as the mutant form and had normal pancreatic total (pro)insulin content [measured as immunoreactive insulin (IRI)]. Fasting glucose levels in these transgenics and in homozygous control mice expressing native human (pro)insulin were normal, although levels were abnormally elevated during ip glucose tolerance testing. In the presence of extracellular calcium, regulated IRI release from the isolated perfused pancreas of the transgenic mice was undetectable in the absence of secretagogues and responded with normal phasic kinetics when stimulated with increasing steps of glucose, with glucose plus isobutylmethylxanthine, or with arginine. Without extracellular calcium (0 calcium plus EGTA), normal pancreas did not release IRI in either the presence or absence of secretagogues. In contrast, without calcium or secretagogues, transgenic pancreas spontaneously and constitutively released IRI at high levels equivalent to those elicited by glucose (22 mM) plus calcium from normal pancreas. This release was partially inhibited by glucose or arginine. Constitutive secretion was acutely sensitive to calcium; inhibition occurred within minutes after the addition of calcium and quickly returned to its characteristic level (with overshoot) when calcium was subsequently removed. Somatostatin, at a concentration that caused 50% inhibition of normal glucose-stimulated secretion, did not affect constitutive release. Control pancreas from the transgenic mice, expressing native human (pro)insulin, responded normally to secretagogues and did not constitutively release hormone in the absence of calcium. It is concluded that expression of monomeric human insulin in pancreatic beta-cells from transgenic mice did not interfere with normal phenotypic insulin secretion, indicating that the functional secretory apparatus was not impaired. Constitutive secretion of IRI from the intact pancreas requires both the expression of a monomeric form of insulin and the absence of extracellular calcium, two conditions that reduce aggregation. These results are consistent with the hypothesis that protein aggregation favors sorting to the regulated pathway, whereas suppressed aggregation causes sorting for constitutive release.

Animals↗