Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating G(q)- and G(i)-linked signaling in the parathyroid, kidney, and intestine. CaR is ubiquitinated by the E3 ligase dorfin and degraded via the endoplasmic reticulum-associated degradation pathway (Huang, Y., Niwa, J., Sobue, G., and Breitwieser, G. E. (2006) J. Biol. Chem. 281, 11610-11617). Here we provide evidence for a conformational or functional checkpoint in CaR biogenesis using two complementary approaches. First we characterized the sensitivity of loss- or gain-of-function CaR mutants to proteasome inhibition by MG132. The stabilization of loss-of-function mutants and insensitivity of gain-of-function mutants to MG132 suggests that receptor sensitivity to calcium influences susceptibility to proteasomal degradation. Second, we used the allosteric activator NPS R-568 and antagonist NPS 2143 to promote the active and inactive conformations of wild type CaR, respectively. Overnight culture in NPS R-568 increased expression of CaR, whereas NPS 2143 had the opposite effect. NPS R-568 and NPS 2143 differentially regulated maturation and cell surface expression of wild type CaR, directly affecting maximal signaling responses. NPS R-568 rescued expression of loss-of-function CaR mutants, increasing plasma membrane expression and ERK1/2 phosphorylation in response to 5 mM Ca(2+). Disorders of calcium homeostasis caused by CaR mutations may therefore result from altered receptor biogenesis independent of receptor function, i.e. a protein folding disorder. The allosteric modulators NPS R-568 and NPS 2143 not only alter CaR sensitivity to calcium and hence signaling but also modulate receptor expression.
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PMID:Rescue of calcium-sensing receptor mutants by allosteric modulators reveals a conformational checkpoint in receptor biogenesis. 1728 38

The seven-spanning calcium-sensing receptor (CaSR) activates multiple G proteins including Gq and Gi, and thereby activates a variety of second messengers and inhibits parathyroid hormone (PTH) secretion. However, the exact signaling mechanisms underlying the functional activity of CaSR are not yet fully understood. The heterozygous inactivation of CaSR or its inhibition by antibody blocking results in either familial hypocalciuric hypercalcemia or acquired hypocalciuric hypercalcemia (AHH), respectively. Here, we report the identification of a unique CaSR autoantibody in an AHH patient. Paradoxically, we find that this autoantibody potentiates the Ca(2+)/Gq-dependent accumulation of inositol phosphates by slightly shifting the dose dependence curve of the Ca(2+) mediated activation of phosphatidylinositol turnover to the left, whereas it inhibits the Ca(2+)/Gi-dependent phosphorylation of ERK1/2 in HEK293 cells stably expressing human CaSR. Treatment of these same cells with a calcimimetic, NPS-R-568, augments the CaSR response to Ca(2+), increasing phosphatidylinositol turnover and ERK1/2 phosphorylation, and overcoming the autoantibody effects. Our observations thus indicate that a calcium-stimulated CaSR primed by a specific autoantibody adopts a unique conformation that activates Gq but not Gi. Our findings also suggest that CaSR signaling may act via both Gq and Gi to inhibit PTH secretion. This is the first report of a disease-related autoantibody that functions as an allosteric modulator and maintains G protein-coupled receptors (GPCRs) in a unique active conformation with its agonist. We thus speculate that physiological modulators may exist that enable an agonist to specifically activate only one signaling pathway via a GPCR that activates multiple signaling pathways.
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PMID:An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations. 1737 16

Accelerated medial calcification is a major cause of premature cardiovascular mortality in patients with chronic kidney disease (CKD). Evidence suggests that extracellular concentration of Ca2+ and vascular smooth muscle cells may play a pivotal role in the pathogenesis of vascular calcification. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that is expressed in a range of tissues, but characterization of its expression and function in the cardiovascular system is limited. Here we report the expression of CaSR mRNA (RT-PCR) and protein (Western blotting and immunocytochemistry) in human aortic smooth muscle cells (HAoSMC). Treatment of HAoSMC with Ca2+ (0-5 mM; 0-30 min) or the CaSR agonists gentamycin and neomycin (0-300 microM; 0-30 min) resulted in a dose- and time-dependent phosphorylation of ERK1/2. Gentamycin- and neomycin-mediated ERK1/2 stimulation was inhibited by pretreatment with PD-98059, an ERK-activating kinase 1 (MEK1) inhibitor, confirming specificity of the observed effects. ERK1/2 activation was inhibited in HAoSMC, with CaSR expression knocked down by transfection with specific small-interference RNA, which confirmed that the observed neomycin/gentamycin-induced MEK1/ERK1/2 activation was mediated via the CaSR. CaSR mRNA and protein were also expressed in large and small arteries from normal subjects (kidney donors) and patients with end-stage renal disease (ESRD). The CaSR was detected in smooth muscle and endothelial cells. Expression was significantly lower in arteries from ESRD patients. In conclusion, these data not only demonstrate the presence of a functional CaSR in human artery but show a correlation between CaSR expression and progression of CKD.
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PMID:Extracellular calcium-sensing receptor is functionally expressed in human artery. 1753 80

Gastrointestinal reflux disease and eosinophilic esophagitis are characterized by basal cell hyperplasia. The extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor, which may be activated by divalent agonists, is expressed throughout the gastrointestinal system. The CaSR may regulate proliferation or differentiation, depending on cell type and tissue. The current experiments demonstrate the expression of the CaSR on a human esophageal epithelial cell line (HET-1A) and the location and expression of the CaSR in the human esophagus. CaSR immunoreactivity was seen in the basal layer of normal human esophagus. CaSR expression was confirmed in HET-1A cells by RT-PCR, immunocytochemistry, and Western blot analysis. CaSR stimulation by extracellular calcium or agonists, such as spermine or Mg(2+), caused ERK1 and 2 activation, intracellular calcium concentration ([Ca(2+)](i)) mobilization (as assessed by microspecfluorometry using Fluo-4), and secretion of the multifunctional cytokine IL-8 (CX-CL8). HET-1A cells transiently transfected with small interfering (si)RNA duplex against the CaSR manifested attenuated responses to Ca(2+) stimulation of phospho- (p)ERK1 and 2, [Ca(2+)](i) mobilization, and IL-8 secretion, whereas responses to acetylcholine (ACh) remained sustained. An inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC) (U73122) blocked CaSR-stimulated [Ca(2+)](i) release. We conclude that the CaSR is present on basal cells of the human esophagus and is present in a functional manner on the esophageal epithelial cell line, HET-1A.
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PMID:The extracellular calcium-sensing receptor (CaSR) on human esophagus and evidence of expression of the CaSR on the esophageal epithelial cell line (HET-1A). 1796 59

Extracellular Ca reduces parathyroid hormone (PTH) levels through several mechanisms, but many details of the intracellular steps involved have been difficult to elucidate because of the lack of a suitable parathyroid cell model. The present studies utilized our Ca-responsive bovine parathyroid organoid culture system (pseudoglands) to examine PTH mRNA in intact parathyroid cells. Increasing medium calcium from 0.4 to 3.0 mM reduced PTH mRNA to 20-30% of basal by 16 h. Reducing medium Ca from 3.0 to 0.4 mM restored PTH mRNA levels over a 24-h period. PTH mRNA was also reduced by the calcimimetic R-568, confirming the role of the calcium-sensing receptor. PTH decay rates were determined by placing pseudoglands in either 0.4 or 3.0 mM Ca for 2 h and then blocking gene transcription. PTH mRNA remained stable for at least 24 h in pseudoglands incubated in 0.4 mM Ca, but fell gradually by 62% in the presence of 3.0 mM Ca. Blocking transcription prior to the addition of high-Ca medium dramatically blunted the Ca-induced degradation of PTH mRNA, indicating that acceleration of PTH mRNA decay by Ca requires gene transcription. Pharmacologic investigation of the signaling pathways involved indicated that the Ca-induced reduction of PTH mRNA did not involve MAP kinase, phospholipase D, or cyclic AMP. However, increasing cytosolic Ca with thapsigargin or the Ca ionophore A23187 decreased PTH mRNA levels. In summary, Ca-mediated destabilization of PTH mRNA requires gene transcription and involves increases in cytosolic Ca.
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PMID:Destabilization of parathyroid hormone mRNA by extracellular Ca2+ and the calcimimetic R-568 in parathyroid cells: role of cytosolic Ca and requirement for gene transcription. 1809 93

Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.
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PMID:Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion. 1845 48

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galphai in MMECs but coupled to Galphas in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.
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PMID:Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells. 1862 40

Calcium-sensing receptor (CaSR) plays key role in vascular calcification in patients with chronic kidney disease (CKD). We investigated the role of CaSR in regulating smooth muscle cell (SMC) proliferation and apoptosis. Incubation with 300 microM neomycin (CaSR agonist) resulted in 7.5-fold (p<0.05) increase in ERK1,2 phosphorylation. It was reduced (p<0.01) by 10 microM PD98059 (MEK1 inhibitor), indicating that CaSR agonist-induced effects were mediated via MEK1/ERK1,2 pathway. ERK1,2 phosphorylation was abolished by 5 microM U73122 (PLC inhibitor), indicating that PLC signalling was crucial for MEK1/ERK1,2 activation. Confirming PLC activation, inositol triphosphate (IP3) production was increased by neomycin/gentamycin (p<0.05) and reduced by U73122. To confirm that ERK1,2 and PLC signalling were mediated via CaSR, Human Aortic SMC (HAoSMC) were transfected with CaSR siRNA. CaSR knockdown resulted in lower ERK1,2 neomycin response and IP3 production (p<0.01). Neomycin increased HAoSMC proliferation >3-fold, which was reduced in CaSR knockdown cells (p<0.01) and further inhibited by PD98059 and U73122 (p<0.05). Apoptosis was not affected by neomycin treatment. U73122 produced 3.5-fold increase in HAoSMC apoptosis, which was further increased by CaSR knockdown (5-fold, p<0.05). In conclusion, stimulation of CaSR leads to activation of MEK1/ERK1,2 and PLC pathways and up-regulation of cell proliferation. CaSR-mediated PLC activation is important for SMC survival and protection against apoptosis.
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PMID:Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis. 1908 23

In patients with chronic renal failure, the heart undergoes remodeling, characterized by hypertrophy, fibrosis, and capillary/myocyte mismatch. In this study, we observed the effects of the calcimimetic agent R-568 on microvascular disease and interstitial fibrosis of the heart. Three-month-old male Sprague-Dawley rats were randomized to subtotal nephrectomy (SNX) or sham operation and subsequently received vehicle or R-568 under two experimental protocols, one for 1 month and the other for 3 months. Echocardiography, capillary length density, volume density of interstitial tissue, and immunohistochemistry and western blots (calcium-sensing receptor, collagen I and III, transforming growth factor (TGF)-beta, mitogen-activated protein kinases, and nitrotyrosine) were assessed. After SNX, weight and wall thickness of the left and the right ventricle were elevated. The ratio of heart to body weight and interventricular septum thickness were not changed by R-568 treatment. The left ventricle fractional shortening (by echocardiography) was lower in SNX; this was ameliorated by R-568. Reduced capillary length density and increased interstitial fibrosis in SNX were improved by R-568, which also reduced the expression of TGF-beta, and collagen I and III. The calcimimetic increased the activation of ERK-1/2, normalized p38 and JNK signaling, and prevented oxidative stress. We conclude that lowering parathyroid hormone with a calcimimetic significantly improves cardiac histology and function but not the left ventricular mass in SNX.
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PMID:Interstitial fibrosis and microvascular disease of the heart in uremia: amelioration by a calcimimetic. 1918 10

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.
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PMID:Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor. 1928 78


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