Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CONSTITUTION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed1997

Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines.

Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak/Stat (Janus kinase/signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3(sm), was found to be constitutively activated, i.e., (i) Stat3(sm) was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (ii) band shift assays and immunoprecipitations of DNA/Stat complexes showed constitutive DNA-binding properties of Stat3(sm); and (iii) Stat3(sm) was constitutively associated with Jak3. The abnormal activation of Stat3(sm) was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3(fm)) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3(sm) and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak/Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient.

Cell Division↗
PubMed1995

SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane domain.

A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.

3T3 Cells↗
PubMed1998

Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha).

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in fatty acid metabolism, levels of peroxisomal and mitochondrial fatty acid metabolizing enzymes were determined in the PPARalpha null mouse. Constitutive liver beta-oxidation of the long chain fatty acid, palmitic acid, was lower in the PPARalpha null mice as compared with wild type mice, indicating defective mitochondrial fatty acid catabolism. In contrast, constitutive oxidation of the very long chain fatty acid, lignoceric acid, was not different between wild type and PPARalpha null mice, suggesting that constitutive expression of enzymes involved in peroxisomal beta-oxidation is independent of PPARalpha. Indeed, the PPARalpha null mice had normal levels of the peroxisomal acyl-CoA oxidase, bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase), and thiolase but lower constitutive expression of the D-type bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase). Several mitochondrial fatty acid metabolizing enzymes including very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-ketoacyl-CoA thiolase, and long chain acyl-CoA synthetase are also expressed at lower levels in the untreated PPARalpha null mice, whereas other fatty acid metabolizing enzymes were not different between the untreated null mice and wild type mice. A lower constitutive expression of mRNAs encoding these enzymes was also found, suggesting that the effect was due to altered gene expression. In wild type mice, both peroxisomal and mitochondrial enzymes were induced by the peroxisome proliferator Wy-14,643; induction was not observed in the PPARalpha null animals. These data indicate that PPARalpha modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid beta-oxidation, thus establishing a role for the receptor in fatty acid homeostasis.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

The human B1 bradykinin receptor exhibits high ligand-independent, constitutive activity. Roles of residues in the fourth intracellular and third transmembrane domains.

The B1 bradykinin (BK) receptor (B1R) is a seven-transmembrane domain, G protein-coupled receptor that is induced by injury and important in inflammation and nociception. Here, we show that the human B1R exhibits a high level of ligand-independent, constitutive activity. Constitutive activity was identified by the increase in basal cellular phosphoinositide hydrolysis as a function of the density of the receptors in transiently transfected HEK293 cells. Several B1R peptide antagonists were neutral antagonists or very weakly efficacious inverse agonists. Constitutive B1R activity was further increased by alanine mutation of Asn(121) in the third transmembrane domain of the receptor (B1A(121)). This mutant resembled the agonist-preferred receptor state since it also exhibited increased agonist affinity and decreased agonist responsiveness. A dramatic loss of constitutive activity occurred when the fourth intracellular C-terminal domain (IC-IV) of the human B2 BK receptor subtype (B2R), which exhibits minimal constitutive activity, was substituted in either B1R or B1A(121) to make B1(B2ICIV) and B1(B2ICIV)A(121), respectively. Activity was partially recovered by subsequent alanine mutation of a cluster of two serines and two threonines in IC-IV of either B1(B2ICIV) or B1(B2ICIV)A(121), a cluster that is important for B2R desensitization. The ligand-independent, constitutive activity of B1R therefore depends on epitopes in both transmembrane and intracellular domains. We propose that the activity is primarily due to the lack of critical epitopes in IC-IV that regulate such activity.

Amino Acid Sequence↗
PubMed2001

Constitutively active mu-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin.

The most convincing evidence demonstrating constitutive activation of mu-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing mu-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.

Adenylate Cyclase Toxin↗
PubMed2003

Identification of an autoregulatory feedback pathway involving interleukin-1alpha in induction of constitutive NF-kappaB activation in pancreatic cancer cells.

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.

Cell Line, Tumor↗
PubMed2004

Constitutive endocytic cycle of the CB1 cannabinoid receptor.

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.

Cell Line↗
PubMed2005

Differential STAT5 signaling by ligand-dependent and constitutively active cytokine receptors.

Many leukemia and cancer cells exhibit constitutive activation of STAT5, which was suggested to provide an anti-apoptotic advantage. Transformation of cytokine-dependent hematopoietic cells, such as Ba/F3 cells to autonomous growth and tumorigenicity equally results in selection for constitutive activation of STAT5. We compared STAT5 signaling between erythropoietin(Epo)-dependent cells and cells that were transformed by oncogenic activation of the erythropoietin receptor (EpoR) by coexpression of the gp55-P envelope protein of the spleen focus forming virus or by expression of the R129C constitutively active EpoR mutant. In transformed cells it was mainly STAT5B that was constitutively activated. In contrast, Epo stimulation activated both STAT5A and STAT5B. In transformed cells, chromatin immunoprecipitation (ChIP) showed STAT5 to be physically bound to promoters of STAT5 target genes, such as Bcl(XL), and to be able to promote transactivation of the Bcl(XL) promoter in a constitutive fashion. Sequencing of native sequences after ChIP with anti-STAT5 antibodies in Epo-dependent and -transformed cells indicated that in gp55-transformed cells, STAT5B bound in the chromatin not only to N3 high affinity, but also to low affinity N4 GAS sites. Transactivation for N3 GAS sites in luciferase reporters was specific to gp55 transformation. Because we also found preferential constitutive STAT5B activation after transformation of cells by a truncated form of the G-CSF-R that produces severe neutropenia (Kostmann syndrome) and favors leukemia in humans, we discuss the potential role of STAT5B in oncogenic transformation of hematopoietic cells.

Active Transport, Cell Nucleus↗
PubMed2004

Interleukin-1 beta gene polymorphism related with allergic pathogenesis in Iris constitution.

Iridological constitution has a strong familial aggregation and is implicated in heredity. The aetiology of inflammatory bowel disease is still unknown. However, from genetic epidemiological studies there is considerable evidence that genetic factors are associated with both Crohn's disease and ulcerative colitis. We investigated the relationships between Iridological constitution and interleukin 1 beta (IL-1beta) gene polymorphism. IL-1beta is a major proinflammatiry cytokine, and the polymorphisms of this gene have been shown to be of importance in a number of diseases. Especially, IL-1 has been suspected of involvement in allergic pathogenesis. Also, IL-1beta genotype is one of the genetic markers of gastric cancer. Therefore, we classified 166 individuals according to Iris constitution, and determined IL-1beta genotype. The frequencies of Iris constitutions as follows: neurogenic type, 41 (24.7%); abdominal connective tissue weakness type, 53 (31.9%); cardio-renal connective tissue weakness type, 50 (30.1%); the others type, 22 (13.3%). Especially, the frequency of abdominal connective tissue weakness type was higher in C/T genotype than in the remaining constitutions although the statistical power was very weak. Furthermore, we first attempted to explore possible involvement of the IL-1beta polymorphism and the Iris constitution.

Complementary Therapies↗
PubMed1995

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations.

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.

Genes↗
PubMed1979

Mating-type effect on cis mutations leading to constitutivity of ornithine transaminase in diploid cells of Saccharomyces cerevisiae.

Cis-acting regulatory mutations have been isolated that affect L-ornithine transaminase (OTAse), an enzyme catalyzing the second step of arginine breakdown in yeast. These mutations lead to constitutive synthesis of OTAse at various levels. Two different types of mutations have been recovered, both of which are tightly linked to the structural gene (cargB) for this enzyme. One type behaves as a classical operator-constitutive mutation similar to the cargB+O---1 mutation previously described (DUBOIS et al. 1978). The second type is peculiar in two respects: the higher level of constitutive OTAse synthesis and the expression of constitutivity in diploid cells. These mutations are designated cargB+Oh. They behave as usual operator-constitutive mutations in diploid strains homozygous for mating type (a/a or alpha/alpha), but the constitutivity is strongly reduced in a/alpha diploid cells.

Diploidy↗
PubMed2006

Extent of constitutive histone H2AX phosphorylation on Ser-139 varies in cells with different TP53 status.

In response to DNA damage by genotoxic agents, histone H2AX is phosphorylated on Ser-139. However, during the cell cycle, predominantly in S and G(2)M phase, histone H2AX is also phosphorylated in untreated normal and tumour cells. This constitutive H2AX phosphorylation is markedly reduced by exposure of cells to the reactive oxygen species scavenger N-acetyl-L-cysteine. Therefore, it appears likely that constitutive H2AX phosphorylation reflects the ongoing oxidative DNA damage induced by the reactive oxygen species during progression through the cell cycle. Because the tumour suppressor p53 (tumour protein p53) is known to induce transcription of genes associated with cell response to oxidative stress, we have compared the intensity of constitutive H2AX phosphorylation, and the effect of N-acetyl-L-cysteine on it, in cells with different tumour protein p53 status. These were human lymphoblastoid cell lines derived from WIL2 cells: TK6, a p53 wt line, NH32, a tumour protein p53 knock-out derived from TK6, and WTK1, a WIL2-derived line that expresses a homozygous mutant of tumour protein p53. Also tested were the tumour protein p53-null promyelocytic HL-60 cells. The degree of constitutive H2AX phosphorylation was distinctly lower in NH32, WTK1 and HL-60 compared to TK6 cells in all phases of the cell cycle. Also, the degree of attenuation of constitutive H2AX phosphorylation by N-acetyl-L-cysteine was less pronounced in NH32, WTK1, and HL-60, compared to TK6 cells. However, the level of reactive oxygen species detected by the cells' ability to oxidize carboxyl-dichlorodihydrofluorescein diacetate was not significantly different in the cell lines studied, which would suggest that regardless of tumour protein p53 status, the level of oxidative DNA damage was similar. The observed higher level of constitutive H2AX phosphorylation in cells harbouring wt tumour protein p53 may thus indicate that tumour protein p53 plays a role in facilitating histone H2AX phosphorylation, an important step in the mobilization of the DNA repair machinery at the site of DNA double-strand breaks.

Acetylcysteine↗
PubMed2005

Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor.

A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT1) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT(1) receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein-coupled receptor (GPCR) activation.

Amino Acid Sequence↗
PubMed2006

Generation of constitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia.

Calpain, a Ca(2+)-dependent cysteine protease, in vitro converts calcineurin (CaN) to constitutively active forms of 45 kDa and 48 kDa by cleaving the autoinhibitory domain of the 60 kDa subunit. In a mouse middle cerebral artery occlusion (MCAO) model, calpain converted the CaN A subunit to the constitutively active form with 48 kDa in vivo. We also confirmed increased Ca(2+)/CaM-independent CaN activity in brain extracts. The generation of constitutively active and Ca(2+)/CaM-independent activity of CaN peaked 2 h after reperfusion in brain extracts. Increased constitutively active CaN activity was associated with dephosphorylation of dopamine-regulated phosphoprotein-32 in the brain. Generation of constitutively active CaN was accompanied by translocation of nuclear factor of activated T-cells (NFAT) into nuclei of hippocampal CA1 pyramidal neurons. In addition, a novel calmodulin antagonist, DY-9760e, blocked the generation of constitutively active CaN by calpain, thereby inhibiting NFAT nuclear translocation. Together with previous studies indicating that NFAT plays a critical role in apoptosis, we propose that calpain-induced CaN activation in part mediates delayed neuronal death in brain ischemia.

Analysis of Variance↗
PubMed2004

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor.

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.

Adenylyl Cyclases↗
PubMed2003

Generation and characterization of highly constitutive active histamine H3 receptors.

Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wild-type (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and Ki values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased Ki values against the MT6 receptors (2.5- to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor.

Animals↗
PubMed2005

High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3.

The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded by equine herpesvirus 2 (EHV2), the DRY motif is substituted with a DTW motif. Nevertheless, this receptor signaled with high constitutive activity through Gi as determined by a receptor-mediated inhibition of forskolin-induced cAMP-production and by an induction of the serum response element-driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor of activated T cells. Coexpression of the ORF74-EHV2 receptor with the promiscuous G protein Gqi4myr supported the constitutive Gi activation as determined by inositol phosphate turnover and CREB activation. The constitutive activity was inhibited by nonpeptide inverse agonists with micromolar potencies, and the chemokine CXCL6 acted as a high-affinity agonist. It is noteworthy that reconstitution of the DRY motif resulted in a 4- to 5-fold decrease of the constitutive activity. Both the wild type and the receptor with the reconstituted DRY motif were expressed at the cell surface as indicated by immunohistochemistry and enzyme-linked immunosorbent assay analysis. It is concluded that the Arg of the DRY motif in transmembrane helix 3 is not essential for G protein coupling based on the constitutive as well as the ligand-mediated activity observed for ORF74-EHV2.

Amino Acid Motifs↗