[New regulations on self-administration and responsibility in constitutional health insurance. 2. Legislative change in constitutional health insurance].
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Attitude of Health Personnel↗
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Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors.
The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.
Animals↗
Skin responses to ultraviolet radiation: effects of constitutive pigmentation, sex, and ancestry.
Constitutive skin pigmentation and skin responses to ultraviolet radiation were measured on a sample of volunteers (n=250) living in State College, PA, USA. The sample was composed of individuals of European American (n=190), Hispanic (n=45), and East Asian ancestry (n=15). Constitutive pigmentation was measured using the Adjusted Melanin Index (AMI), Erythemal Dose Response (EDR) was measured using the slope of a* at 24 h (Deltaa*), and Melanogenic Dose-Response (MDR) was measured using DeltaAM, the slope of AMI at 7 d. The relationships between constitutive skin pigmentation, EDR, MDR, sex, age, and ancestry were investigated. European Americans showed a lower constitutive pigmentation, had a significantly higher burn response (EDR), and had a significantly lower tanning response (MDR) than Hispanics and East Asians. No significant difference is seen between Hispanics and East Asians for either constitutive pigmentation or EDR. Constitutive pigmentation in females was slightly lower than in males in all three samples, but the difference was not significant. While no differences were observed in MDR between sexes, males had a stronger EDR than females regardless of population or constitutive pigmentation level, and this difference was significant in European Americans and Hispanics. We observed no age-related differences in any of the populations or measures investigated. We evaluated the relationship between constitutive pigmentation, EDR and MDR. There was a strong inverse correlation between constitutive pigmentation and EDR in the three samples (European Americans, R2=0.176, P < 0.001; Hispanics, R2=0.204, P=0.009; East Asians, R2=0.223, P=0.098) and a strong direct correlation between constitutive pigmentation and MDR in European Americans and Hispanics (European Americans, R2=0.094, P < 0.001; Hispanics, R2=0.164, P=0.012). In other words, persons with lower constitutive pigmentation both burn more and tan less than persons with higher pigmentation. However, after controlling for constitutive pigmentation, EDR and MDR were significantly correlated in European Americans (R2=0.041 P=0.006). Thus, the general observation that persons who burn more tan less is probable because of the common link that these two phenotypes have with constitutive skin pigmentation and, in fact, once pigmentation has been adjusted for, there is a positive correlation between tanning response and burning response in European Americans.
Adolescent↗
Characteristics of cardiac myxoma with constitutional signs: a multicenter study in Japan.
BACKGROUND: A relationship between constitutional signs in patients with cardiac myxoma and interleukin-6 has been noted. However, there is little information about characteristics of cardiac myxomas associated with constitutional signs. HYPOTHESIS: The objective of this study was to clarify the characteristics of myxoma patients who had constitutional signs. METHODS: Questionnaires were sent to cardiology or cardiovascular surgery divisions at university hospitals throughout Japan. Constitutional signs were considered present when a patient had fever, weight loss, or elevations of C-reactive protein or gammaglobulin. In addition, interleukin-6 concentrations were evaluated in some patients. RESULTS: Data were obtained in 249 patients with primary cardiac tumors (204 myxomas, 15 other primary benign tumors, and 30 primary malignant tumors), confirmed histologically between 1993 and 1996. Fever and weight loss were observed in 15 and 6% of patients with myxoma, respectively, while C-reactive protein and gammaglobulin were increased in 39 and 21%, respectively. This amounted to a prevalence of constitutional signs in 49%. All constitutional signs disappeared after tumor resection. Age, gender, tumor site, and frequency of thrombosis did not differ between patients with and without constitutional signs. Tumors associated with constitutional signs were significantly more likely to be large, multiple, or recurrent than those unassociated with constitutional signs. CONCLUSIONS: Constitutional signs are present in about half of patients with myxoma. Large or multicentric tumors are likely to induce constitutional signs, which are reversible upon resection. These might suggest that constitutional signs result when interleukin-6 concentrations exceed a certain threshold.
Adult↗
Constitutive nuclear NF-kappa B in cells of the monocyte lineage.
In monocytes, the nuclear factor NF-kappa B has been invoked as an important transcription factor in the expression of cytokine genes, of cell-surface receptors and in the expression of human immunodeficiency virus. In such cells, DNA binding activity of NF-kappa B can be detected without intentional stimulation. In our studies, cells of the human monocytic line Mono Mac 6, cultured in medium containing fetal-calf serum and low levels of lipopolysaccharide (LPS), also exhibit such 'constitutive' NF-kappa B, as demonstrated by mobility-shift analysis of nuclear extracts. This nuclear NF-kappa B was still present when contaminant LPS was removed by ultrafiltration and when serum was omitted. Protein-DNA complexes of constitutive NF-kappa B are similar in mobility to the LPS-induced NF-kappa B and both are recognized by an antibody specific to the p50 subunit of NF-kappa B. By contrast, treatment of cells with pyrrolidine dithiocarbamate (PDTC) will only block LPS-induced NF-kappa B, but not the constitutive binding protein. Using LPS-free and serum-free conditions, constitutive NF-kappa B can be detected in different cell lines of the monocytic lineage (HL60, U937, THP-1, Mono Mac 1 and Mono Mac 6), but not in Molt 4 T cells or K562 stem cells. When ordered according to stage of maturation, the amount of constitutive NF-kappa B was not increased in more mature cell lines. Furthermore, when inducing differentiation in Mono Mac 6 cells, with vitamin D3, no change in constitutive or inducible NF-kappa B can be detected. Analysis of primary cells revealed substantial constitutive NF-kappa B-binding activity in blood monocytes, pleural macrophages and alveolar macrophages. The constitutive NF-kappa B appears to be functionally active, since a low level of tumour necrosis factor (TNF) transcript is detectable in monocytes, and this level can be increased by blocking transcript degradation using cycloheximide. The level of constitutive NF-kappa B in these cells is variable and is frequently found to be lower in the more mature macrophages. Constitutive NF-kappa B was not maintained by autocrine action of cytokines TNF, interleukin 6, interleukin 10, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor, since neutralizing antibodies did not reduce constitutive DNA-binding activity. Furthermore, blockade of prostaglandin or leukotriene biosynthesis did not affect constitutive NF-kappa B.(ABSTRACT TRUNCATED AT 400 WORDS)
Base Sequence↗
[Superior mesenteric lymph nodes in adult humans of different body constitution types].
The number of superior mesenteric lymph nodes regional for small intestine and its mesentery ranges from 66 to 232 in adults with brachymorphic type of constitution, from 85 to 404 in those with mesomorphic type of constitution and from 105 to 327 in adults with dolichomorphic type of constitution. Average lymph node number equals 159 in subjects with brachymorphic type of constitution and it is 212 and 175 in subjects with dolichomorphic and mesomorphic types of constitution respectively. Dimensions of lymph nodes studied varied from 1 x 1 x 1 to 52 x 10 x 5 mm in adults with brachymorphic type of constitution, from 1 x 1 x 1 to 40 x 8 x 4 mm in those with mesomorphic type of constitution and from 1 x 1 x 1 to 38 x 8 x 4 mm. Average values of node cross dimensions were 7.1, 6.2 and 5.3 mm respectively. Consequently, superior mesenteric lymph nodes are comparatively numerous in adults with dolichomorphic type of constitution, while node dimensions, on the contrary, are highest in adults with brachymorphic type of constitution. Rounded and oval nodes were encountered predominantly in adults with brachymorphic constitution, while segmented and ribbon-like--in people with brachymorphic type of constitution.
Adult↗
Three different stable human breast adenocarcinoma sublines that overexpress ALDH3A1 and certain other enzymes, apparently as a consequence of constitutively upregulated gene transcription mediated by transactivated EpREs (electrophile responsive elements) present in the 5'-upstream regions of these genes.
ALDH3A1 catalyzes the detoxification of cyclophosphamide, mafosfamide, 4-hydroperoxycyclophosphamide and other oxazaphosphorines. Constitutive ALDH3A1 levels, as well as those of certain other drug-metabolizing enzymes, e.g. NQO1 and CYP1A1, are relatively low in cultured, relatively oxazaphosphorine-sensitive, human breast adenocarcinoma MCF-7 cells. However, transient cellular insensitivity to the oxazaphosphorines can be brought about in these cells by transiently elevating ALDH3A1 levels in them as a consequence of transient exposure to: (1) electrophiles such as catechol that induce the transcription of a battery of genes, e.g. ALDH3A1 and NQO1, having in common an electrophile responsive element (EpRE) in their 5'-upstream regions; or (2) Ah-receptor agonists, e.g. indole-3-carbinol and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, that induce the transcription of a battery of genes, e.g. ALDH3A1, NQO1 and CYP1A1, having in common a xenobiotic responsive element (XRE) in their 5'-upstream regions. Further, MCF-7 sublines that are constitutively, i.e. when grown in the absence of the original selecting pressure, relatively oxazaphosphorine-insensitive as a consequence of constitutively relatively elevated cellular ALDH3A1 levels evolved when MCF-7 cells were: (1) continuously exposed for several months to gradually increasing concentrations of 4-hydroperoxycyclophosphamide or benz(a)pyrene; or (2) briefly exposed (once for 30 min) to a high concentration (1 mM) of mafosfamide. Each of these three stable sublines is constitutively relatively cross-insensitive to benz(a)pyrene and other polycyclic aromatic hydrocarbons. Cellular levels of NQO1, but not of CYP1A1, are also constitutively relatively elevated in each of the three sublines. RT-PCR-based experiments established that ALDH3A1 mRNA levels are constitutively elevated ( approximately 5- to 8-fold) in each of the three sublines. The elevated ALDH3A1 mRNA levels are not the consequence of gene amplification, hypomethylation of a relevant regulatory element, or ALDH3A1 mRNA stabilization. Collectively, these observations suggest that constitutively elevated levels of ALDH3A1 and certain other enzymes in the three stable sublines are probably the consequence of a constitutive change in the cellular concentration of a key component of the EpRE signaling pathway, such that the cellular concentration of the relevant ultimate transactivating factor is constitutively elevated, i.e. gene transcription promoted by transactivated EpREs is constitutively upregulated. Further, constitutively upregulated gene transcription mediated by transactivated EpREs can be relatively easily induced, whereas that mediated by transactivated XREs cannot, at least in MCF-7 cells. Still further, the three sublines may facilitate study of the signaling pathway that leads to transactivation of the EpREs present in the 5'-upstream regions of ALDH3A1, NQO1 and other gene loci.
Aldehyde Dehydrogenase↗
[Cold constitution: analysis of the questionnaire].
"Hie-sho" means having a cold constitution. The authors studied basic data for cold constitution with a questionnaire and with the Cornell Medical Index. The subjects were 318 females, and the average age was 26.4 +/- 6.1 (Mean +/- SD) and the range was 20 approximately 51 years of age. The results were as follows: (1) 38.7% of the subjects had been conscious of having a cold constitution, and it was recognized that the subjects with a cold constitution are found not only in the climacterium but also in the young. (2) The average age of onset of cold constitution was 19.3 +/- 5.1 years old. (3) The parts of the body that usually felt cold were first the feet and then the hands. (4) In winter or at bedtime the cold sensation was increased. And it is characteristic that the sensation was changed by weather or by body condition, especially by the stimulation of coldness. (5) Most subjects with a cold constitution took care in their daily life, but very few of them were treated. (6) Many other physical symptoms were recognized in subjects with a cold constitution, and their CMI scores were high. Therefore these data may be regarded as indicating that most cases of cold constitution are a partial symptom of vegetative disturbance (autonomic nerve dysfunction). (7) Most of the mothers of subjects with a cold constitution also had a cold constitution, which suggests the involvement of a hereditary factor concerning in the development of a cold constitution.
Adult↗
Constitutional genomic instability, chromosome aberrations in tumor cells and retinoblastoma.
Although retinoblastoma (Rb) is initiated as a result of biallelic inactivation of the RB1 gene, additional genetic events (M3) in tumor cells are indicative of their role in the full transformation of retinal cells. We investigated the constitutional genetic instability by fragile site (FS) expression studies and checked its relationship with loci of tumor cytogenetics in a series of 36 retinoblastoma patients (34 nonfamilial and 2 familial cases). Tumor cytogenetics revealed -13/+13, del/t(13)(q14) (50%), +1/del/t(1p/q) (65%), +6/i(6p) (60%), and del(16)(q13)/(q22 approximately q23) (60%). Conventional cytogenetics in leukocytes revealed constitutional del(13q14) in five unilateral Rb (URB) and one trilateral Rb (TRB). Constitutional del(16)(q22) and t(6;12) were also identified in two cases. Constitutional FS analysis showed a significant increase in the cellular fragility, with high prevalence at 13q14, 3p14, 6p23, 16q22 approximately q23, and 13q22 loci in retinoblastoma patients (P<0.05). Patients with constitutional del(13)(q14) demonstrated higher fragility than those with normal constitution. A strong correlation between loci of constitutional FSs and loci of recurrent chromosomal abnormalities in tumors strengthen and support the proposal that FS loci present as inherent genomic instability in retinoblastoma. The chromosomal changes and resultant genetic mutations, along with RB1 mutation events, probably contribute synergistically to the development and progression of Rb malignancy. Implementation of fluorescence in situ hybridization to nonfamilial Rb on a large scale (113 cases) could detect constitutional RB1 deletion in 12.3% of cases, with equally higher incidence in URB (14.7%) and bilateral Rb (13.6%), demonstrating that the true prevalence of patients with predisposition to RB1 mutation in sporadic URB is definitely higher in our populations. Also, higher incidence of constitutional RB1 deletion mosaicism in unilateral than in bilateral Rb indicates that the constitutional genetic mosaicism in URB should be given serious consideration during genetic counseling.
Adolescent↗
Constitutive activity of human prostaglandin E receptor EP3 isoforms.
1. The human EP3 prostaglandin receptor is a seven transmembrane, G protein-coupled receptor that couples to inhibition of adenylyl cyclase. The receptor occurs as at least six isoforms which result from alternative splicing. The isoforms are identical over the first 359 amino acids, comprising the seven transmembrane helices, but differ in the carboxyl terminal tail which ranges in length from 6 to 65 amino acids beyond the common region. 2. We have stably expressed in CHO-K1 cells four of the isoforms (EP3I-EP3IV) and a form of the EP3 receptor (T-359) truncated at the carboxyl-terminal region defined by the alternative splicing site at amino acid number 359. 3. Isoforms EP3I and EP3II showed concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase in CHO-K1 cells by the EP3 receptor agonist, sulprostone. The IC50 calculated for sulprostone inhibition was 0.2 nM for EP3I and 0.15 nM for EP3II. The maximum extent of inhibition was 80% for both isoforms. 4. Isoforms EP3III and EP3IV showed marked constitutive activity, inhibiting forskolin-stimulated adenylyl cyclase in the absence of agonist. EP3IV also displayed some agonist-dependent inhibition whereas EP3III was fully constitutively active. 5. The truncated receptor T-359 was fully constitutively active, inhibiting forskolin-stimulated adenylyl cyclase by about 70% in the absence of agonist, and showed no agonist-dependent inhibition, in agreement with a similar truncation of the mouse EP3 receptor. 6. To confirm that differences in cyclic AMP level between isoforms represent constitutive activity, we treated cells with pertussis toxin for 6 h to abolish Gi function. Pertussis toxin reversed sulprostone-mediated inhibition of cyclic AMP formation in EP3I and EP3II and abolished constitutive activity of EP3III, EP3IV and T-359 so that the level of forskolin-stimulated cyclic AMP produced was the same in all cells and similar to that obtained in mock-transfected cells. In mock-transfected cells, sulprostone had no effect on forskolin-stimulated cyclic AMP formation. 7. For these experiments we chose clones that showed similar expression levels of each isoform, as determined by binding of [3H]-prostaglandin E2 (PGE2) (EP3I, 0.71; EP3II, 1.47; EP3IV, 1.59 pmol mg-1 protein). Mock-transfected cells showed no detectable binding of [3H]-PGE2. In addition, we performed a detailed study of the effects of expression level on constitutive activity. Over a six fold range of expression there was no change in the properties of each isoform with regard to whether it was constitutively active or not. 8. The degree of constitutive activity correlated with the inverse of the length of the C-terminal tail of the isoforms. However, no correlation was found between isoforms from human and mouse: whereas EP3II shows no constitutive activity, its mouse homologue, EP3 gamma, shows almost complete constitutive activity, even though the C-terminal domains of the receptors following the splice site differ in only 7 of 29 amino acids.
Adenylate Cyclase Toxin↗
Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in human head and neck squamous cell carcinoma.
Constitutively activated nuclear factor-kappaB (NF-kappaB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-kappaB in a series of HNSCC cell lines. We confirmed that NF-kappaB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-kappaB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappaB (IkappaBalpha), the natural inhibitor of NF-kappaB. Constitutive NF-kappaB activation in HNSCC was found to be due to constitutive activation of IkappaBalpha kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IkappaBalpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappaB activation. The expression of constitutively active NF-kappaB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappaB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-beta-activated kinase 1 (TAK1), DN-kappa-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-kappaB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappaB and its ligand in HNSCC cells but not interleukin (IL)-1beta. All HNSCC cell lines expressed IL-6, a NF-kappaB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappaB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappaB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.
Carcinoma, Squamous Cell↗
Molecular regulation of constitutive expression of interleukin-8 in human pancreatic adenocarcinoma.
Recent studies have shown that interleukin-8 (IL-8) plays an important role in the growth and metastasis of human pancreatic cancer. In the present study, we determined the molecular regulation of constitutive IL-8 expression in human pancreatic cancer cells. Various human pancreatic cancer cell lines were incubated in vitro. Sixty-seven percent of the cell lines constitutively secreted high levels of IL-8, as determined using enzyme-linked immunosorbent assay. Consistently, these cells constitutively expressed high levels of IL-8 mRNA, as determined using Northern blot analysis. To determine the mechanisms of the high steady-state levels of IL-8 mRNA, the IL-8 half-life and transcription rate were measured. There was no significant difference in IL-8 half-life between cells expressing high and low levels of IL-8. However, higher transcription rates and increased IL-8 promoter activity were observed in the cells constitutively expressing high levels of IL-8. Detailed IL-8 promoter analysis using deletion mutation revealed that the region from -85 to -133 bp was essential for the constitutive IL-8 promoter activity. Also, point-mutation analysis indicated that mutation of NF-kappaB, AP-1, or NF-IL-6 binding sites significantly reduced or eliminated the constitutive IL-8 promoter activity. Consistent with the constitutive IL-8 transcription activity, high levels of constitutive NF-kappaB and AP-1 activity were detected in the cells overexpressing IL-8, as determined using electrophoretic mobility shift assay. In addition, transfection of a dominant-negative I-kappaBalpha expression vector (I-kappaBalphaM) inhibited constitutive NF-kappaB activity and IL-8 expression in pancreatic cancer cells. Collectively, our data demonstrated that constitutive NF-kappaB and AP-1 activation contributes to the overexpression of IL-8, which in turn plays an important role in tumor angiogenesis and contributes to the aggressive biology of human pancreatic cancer.
Adenocarcinoma↗
Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1.
Extracellular signal-regulated kinase (ERK) is an important intermediate in signal transduction pathways that are initiated by many types of cell surface receptors. It is thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Constitutive activation of ERK in fibroblasts elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find factors contributing to constitutive ERK activation. First, we showed that ERK and MEK were constitutively activated in acute leukemias by in vitro kinase assay and immunoblot analysis. However, in only one half of the studied samples, the pattern of ERK activation was similar to that of MEK activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed hyperexpression of ERK in a majority of acute leukemias. In 17 of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression was similar to that of ERK activation. The fact of constitutive activation of ERK in acute leukemias suggested to us the possibility of an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in hematopoietic tissue and known to be upregulated at the transcription level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow (BM) samples showing minimal or no ERK activation (P =.002). Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P =.002). Finally, by further analysis of 26 cases, we showed that a complementary role of MEK activation, ERK hyperexpression, and PAC1 downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is constitutively activated in a majority of acute leukemias, and in addition to the activation of MEK, the hyperexpression of ERK and downregulation of PAC1 also contribute to constitutive ERK activation in acute leukemias.
Calcium-Calmodulin-Dependent Protein Kinases↗
Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA).
The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.
Alanine Transaminase↗
The human formyl peptide receptor as model system for constitutively active G-protein-coupled receptors.
According to the two-state model of G-protein-coupled receptor (GPCR) activation, GPCRs isomerize from an inactive (R) state to an active (R*) state. In the R* state, GPCRs activate G-proteins. Agonist-independent R/R* isomerization is referred to as constitutive activity and results in an increase in basal G-protein activity, i.e. GDP/GTP exchange. Agonists stabilize the R* state and further increase, whereas inverse agonists stabilize the R state and decrease, basal G-protein activity. Constitutive activity is observed in numerous wild-type GPCRs and disease-causing GPCR mutants with increased constitutive activity. The human formyl peptide receptor (FPR) exists in several isoforms (FPR-26, FPR-98 and FPR-G6) and activates chemotaxis and cytotoxic cell functions of phagocytes through G(i)-proteins. Studies in HL-60 leukemia cell membranes demonstrated inhibitory effects of Na(+) and pertussis toxin on basal G(i)-protein activity, suggesting that the FPR is constitutively active. However, since HL-60 cells express several constitutively active chemoattractant receptors, analysis of constitutive FPR activity was difficult. Sf9 insect cells do not express chemoattractant receptors and G(i)-proteins and provide a sensitive reconstitution system for FPR/G(i)-protein coupling. Such expression studies showed that FPR-26 is much more constitutively active than FPR-98 and FPR-G6 as assessed by the relative inhibitory effects of Na(+) and of the inverse agonist cyclosporin H on basal G(i)-protein activity. Site-directed mutagenesis studies suggest that the E346A exchange in the C-terminus critically determines dimerization and constitutive activity of FPR. Moreover, N-glycosylation of the N-terminus seems to be important for constitutive FPR activity. Finally, we discuss some future directions of research.
Amino Acid Sequence↗
Constitutive receptor systems for drug discovery.
This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.
Animals↗
Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors.
Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.
Animals↗
Ligand-induced TCR down-regulation is not dependent on constitutive TCR cycling.
TCR internalization takes place both in resting T cells as part of constitutive TCR cycling, after PKC activation, and during TCR triggering. It is still a matter of debate whether these pathways represent distinct pathways. Thus, some studies have indicated that ligand-induced TCR internalization is regulated by mechanisms distinct from those involved in constitutive internalization, whereas other studies have suggested that the ligand-induced TCR internalization pathway is identical with the constitutive pathway. To resolve this question, we first identified requirements for constitutive TCR cycling. We found that in contrast to PKC-induced TCR internalization where both CD3gamma-S(126) and the CD3gamma leucine-based internalization motif are required, constitutive TCR cycling required neither PKC nor CD3gamma-S(126) but only the CD3gamma leucine-based motif. Having identified these requirements, we next studied ligand-induced internalization in cells with abolished constitutive TCR cycling. We found that ligand-induced TCR internalization was not dependent on constitutive TCR internalization. Likewise, constitutive internalization and recycling of the TCR were independent of an intact ligand-induced internalization of the TCR. In conclusion, ligand-induced TCR internalization and constitutive cycling of the TCR represents two independent pathways regulated by different mechanisms.
Amino Acid Motifs↗