The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.     

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.     

Functions and roles of the extracellular Ca2+-sensing receptor in the gastrointestinal tract

The gastrointestinal tract is vital to food digestion and nutrient absorption as well as normal salt and water homeostasis. Studies over the last several years have shown that the Ca2+-sensing receptor is expressed along the entire gastrointestinal tract. The potential roles for the receptor in gastrointestinal biology are now only beginning to be elucidated and much work remains. Well-studied physiological effects include regulation of gastric acid secretion and modulation of fluid transport in the colon. It remains to be determined if the Ca2+-sensing receptor is involved in calcium handling by the gastrointestinal tract. The ability of organic nutrient receptor agonists/allosteric modifiers, such as polyamines and L-amino acids, to activate the Ca2+-sensing receptor suggest potential roles in signalling nutrient availability to gastric and intestinal epithelial cells. In addition, polyamines are crucial for normal cell proliferation and differentiation required to sustain the rapid turnover of gastrointestinal epithelial cells and the Ca2+-sensing receptor may be involved in this function. Activation of the colonic Ca2+-sensing receptor can abrogate cyclic nucleotide-mediated fluid secretion suggesting a role for the receptor in modifying secretory diarrheas like cholera. Finally, the Ca2+-sensing receptor has been suggested to provide a mechanism for the effect of calcium intake in reducing the risk of colon cancer.     

G protein-coupled, extracellular Ca2+ (Ca o 2+ )-sensing receptor enables Ca o 2+ to function as a versatile extracellular first messenger

The cloning of a G protein-coupled, extracellular Ca²⁺ (Ca _o ²⁺ )-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca _o ²⁺ on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca _o ²⁺ metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca²⁺, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca²⁺ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.     

A double mutation in the extracellular Ca2+-sensing receptor's venus flytrap domain that selectively disables L-amino acid sensing

The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca(2+) release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCgamma1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCgamma1. IL-1-induced phosphorylation of PLCgamma1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCgamma1, indicating mutually dependent interactive roles for Ca(2+), SHP-2, and PLCgamma1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCgamma1 and thereby enhances IL-1-induced Ca(2+) release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation.     

Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0-40 min) of LLCP-K(1) cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression.     

Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.     

小凹蛋白-1下调人脐静脉内皮细胞外钙敏感受体介导的钙内流

为了探讨小凹蛋白-1(caveolin-1,Cav-1)在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)细胞外钙敏感受体(extracellular Ca2+-sensing receptor,CaR)介导Ca2+内流中的作用,本实验研究了细胞膜穴样凹陷(caveolae)结构破坏剂Filipin或Cav-1基因沉默后对CaR介导Ca2+内流的影响。Fura-2/AM负载检测细胞内Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)。结果显示,HUVECs中CaR对不同浓度细胞外Ca2+刺激无反应。无论细胞外为零钙液或含钙液时,精胺(Spermine,2mmol/L)刺激CaR时均引起[Ca2+]i升高(P<0.05),其中细胞外液为含钙液时,[Ca2+]i升高较细胞外为零钙液时更明显(P<0.05),CaR的负性变构调节剂Calhex231(1μmol/L)均可完全阻断Spermine刺激引起的[Ca2+]i升高(P<0.05);相反,Spermine升高[Ca2+]i作用可被Filipin(1.5μ...     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

Real time measurements of water flow in amphibian gastric glands: modulation via the extracellular Ca2+-sensing receptor

The mechanisms for the formation of the osmotic gradient driving water movements in the gastric gland and its modulation via the extracellular Ca(2+)-sensing receptor (CaR) were investigated. Real time measurements of net water flux in the lumen of single gastric glands of the intact amphibian stomach were performed using ion-selective double-barreled microelectrodes. Water movement was measured by recording changes in the concentration of impermeant TEA(+) ions ([TEA(+)](gl)) with TEA(+)-sensitive microelectrodes inserted in the lumen of individual gastric glands. Glandular K(+) (K(+)(gl)) and H(+) (pH(gl)) were also measured by using K(+)- and H(+)-sensitive microelectrodes, respectively. Stimulation with histamine significantly decreased [TEA](gl), indicating net water flow toward the gland lumen. This response was inhibited by the H(+)/K(+)-ATPase inhibitor, SCH 28080. Histamine also elicited a significant and reversible increase in [K(+)](gl) that was blocked by chromanol 293B, a blocker of KCQN1 K(+) channels. Histamine failed to induce net water flow in the presence of chromanol 293B. In the "resting state," stimulation of CaR with diverse agonists resulted in significant increase in [TEA](gl). CaR activation also significantly reduced histamine-induced water secretion and apical K(+) transport. Our data validate the strong link between histamine-stimulated acid secretion and water transport. We also show that cAMP-dependent [K(+)](gl) elevation prior to the onset of acid secretion generates the osmotic gradient initially driving water into the gastric glands and that CaR activation inhibits this process, probably through reduction of intracellular cAMP levels.     

beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor

Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.     

Silencing of filamin A gene expression inhibits Ca2+ -sensing receptor signaling

Filamin plays an important role in actin cytoskeleton organization, membrane stabilization, and anchoring of transmembrane proteins. Using short interfering RNA (siRNA) to selectively target the filamin A gene and silence its expression, we studied the role of filamin A in G protein coupled receptor (GPCR) signaling. Silencing of filamin A protein expression was determined by immunoblotting and immunofluorescence. Functional consequences of filamin A gene silencing were measured by studying its role in MAPK signaling pathways activated by the Ca2+ -sensing receptor. This work defines filamin A involvement in GPCR signaling pathways and describes an additional method for studying its function.     

Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of beta-arrestin to the receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.     

Brevetoxin activation of voltage-gated sodium channels regulates Ca dynamics and ERK1/2 phosphorylation in murine neocortical neurons

Voltage-gated sodium channels (VGSC) are involved in the generation of action potentials in neurons. Brevetoxins (PbTx) are potent allosteric enhancers of VGSC function and are associated with the periodic 'red tide' blooms. Using PbTx-2 as a probe, we have characterized the effects of activation of VGSC on Ca(2+) dynamics and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling in neocortical neurons. Neocortical neurons exhibit synchronized spontaneous Ca(2+) oscillations, which are mediated by glutamatergic signaling. PbTx-2 (100 nm) increased the amplitude and reduced the frequency of basal Ca(2+) oscillations. This modulatory effect on Ca(2+) oscillations produced a sustained rise in ERK1/2 activation. At 300 nm, PbTx-2 disrupted oscillatory activity leading to a sustained increase in intracellular Ca(2+) ([Ca(2+)](i)) and induced a biphasic, activation followed by dephosphorylation, regulation of ERK1/2. PbTx-2-induced ERK1/2 activation was Ca(2+) dependent and was mediated by Ca(2+) entry through manifold routes. PbTx-2 treatment also increased cAMP responsive element binding protein (CREB) phosphorylation and increased gene expression of brain-derived neurotrophic factor (BDNF). These findings indicate that brevetoxins, by influencing the activation of key signaling proteins, can alter physiologic events involved in survival in neocortical neurons, as well as forms of synaptic plasticity associated with development and learning.