Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel activated by oxidative stress or tumor necrosis factoralpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca(2+). We explored whether calmodulin (CaM) is the Ca(2+) sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaM(MUT)) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H(2)O(2) or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([Ca(2+)](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaM(MUT) with TRPM2 dramatically inhibited the increase in [Ca(2+)](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaM(MUT) with TRPM2, and the Ca(2+) dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H(2)O(2). The increase in [Ca(2+)](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca(2+) entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.     

Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C

The serotonin 5-HT(1A) receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT(1A) receptor signaling. We have found that the ubiquitous Ca(2+)-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT(1A) receptor in Chinese hamster ovary fibroblasts. The human 5-HT(1A) receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca(2+)-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K(D) of approximately 110 nm for the i3N peptide and approximately 700 nm for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 +/- 23 nm and 1.70 +/- 0.16 microm. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT(1A) receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT(1A) receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT(1A) receptor phosphorylation and desensitization. These data suggest that the 5-HT(1A) receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.     

Identification of apocalmodulin and Ca2+-calmodulin regulatory domain in skeletal muscle Ca2+ release channel, ryanodine receptor

Fusion proteins and full-length mutants were generated to identify the Ca(2+)-free (apoCaM) and Ca(2+)-bound (CaCaM) calmodulin binding sites of the skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1). [(35)S]Calmodulin (CaM) overlays of fusion proteins revealed one potential Ca(2+)-dependent (aa 3553-3662) and one Ca(2+)-independent (aa 4302-4430) CaM binding domain. W3620A or L3624D substitutions almost abolished completely, whereas V3619A or L3624A substitutions reduced [(35)S]CaM binding to fusion protein (aa 3553-3662). Three full-length RyR1 single-site mutants (V3619A,W3620A,L3624D) and one deletion mutant (Delta4274-4535) were generated and expressed in human embryonic kidney 293 cells. L3624D exhibited greatly reduced [(35)S]CaM binding affinity as indicated by a lack of noticeable binding of apoCaM and CaCaM (nanomolar) and the requirement of CaCaM (micromolar) for the inhibition of RyR1 activity. W3620A bound CaM (nanomolar) only in the absence of Ca(2+) and did not show inhibition of RyR1 activity by 3 microm CaCaM. V3619A and the deletion mutant bound apoCaM and CaCaM at levels compared with wild type. V3619A activity was inhibited by CaM with IC(50) approximately 200 nm, as compared with IC(50) approximately 50 nm for wild type and the deletion mutant. [(35)S]CaM binding experiments with sarcoplasmic reticulum vesicles suggested that apoCaM and CaCaM bind to the same region of the native RyR1 channel complex. These results indicate that the intact RyR1 has a single CaM binding domain that is shared by apoCaM and CaCaM.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.     

Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.     

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献   

A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients

D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

Phosphorylation determines the calmodulin-mediated Ca2+ response and water permeability of AQP0

In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.     

Cyclophane and acyclic cyclophane: novel channel blockers of N-methyl-D-aspartate receptor

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.     

Prostaglandin receptors: advances in the study of EP3 receptor signaling

Prostaglandin (PG) E(2) produces a broad range of physiological and pharmacological actions in diverse tissues through specific receptors on plasma membranes for maintenance of local homeostasis in the body. PGE receptors are divided into four subtypes, EP1, EP2, EP3, and EP4, which have been identified and cloned. These EP receptors are members of the G-protein coupled receptor family. Among these subtypes, the EP3 receptor is unique in its ability to couple to multiple G proteins. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via G(i) activation, and in Ca(2+)-mobilization through G(beta)(gamma) from G(i). Along with G(i) activation, the EP3 receptor can stimulate cAMP production via G(s) activation. Recent evidence indicates that the EP3 receptor can augment G(s)-coupled receptor-stimulated adenylyl cyclase activity, and can also be coupled to the G(13) protein, resulting in activation of the small G protein Rho followed by morphological changes in neuronal cells. This article focuses on recent studies on the novel pathways of EP3 receptor signaling.     

Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.     

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle

Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a direct contractile response. In contrast, M(2) receptors interact with G(i) and G(o) to inhibit adenylyl cyclase and Ca(2+)-activated K(+) channels and to potentiate a Ca(2+)-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M(2) receptor on contraction should be conditional upon mobilization of Ca(2+) by another receptor such as the M(3). Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M(2) and M(3) receptors should resemble the profile of the directly acting receptor (i.e., the M(3)) and not that of the conditionally acting receptor (i.e., the M(2)). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M(2) receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M(3) receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M(2) and M(3) receptors.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.     

Calmodulin binding to the Fas death domain. Regulation by Fas activation

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.