Ligand-specific conformation of extracellular loop-2 in the angiotensin II type 1 receptor.

The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor.

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.

Modulation of peptidyl arginine deiminase 2 and implication for neurodegeneration.

PURPOSE: To demonstrate that elevated pressure increases the peptidyl arginine deiminase 2 (PAD2) expression in cultured astrocytes in vitro that can be modulated by pharmacological agents modulating intracellular calcium. METHODS: Isolated rat brain astrocytes were subjected to pressure treatment. Western and immunohistochemical analyses detected PAD2 protein expression. Calcium measurements were achieved employing fluorescence-based microscopic imaging and quantification system. Experiments were repeated with human optic nerve head-derived astrocytes. RESULTS: PAD2 has recently been shown to be associated with glaucomatous optic nerve. Astrocytes subjected to pressure (25-100 mmHg) show elevated level of PAD2, increased intracellular calcium, and concomitant citrullination but not significant cell death. PAD2 expression in response to elevated pressure may play a role in glaucomatous neurodegeneration. Pressure-treated astrocytes were also subjected to thapsigargin (50-250 nM) treatment, but it is unclear whether this had any further effect in increasing PAD2 expression. Conversely, treatment with calcium chelating agent BAPTA-AM (50-250 nM) results in decreased intracellular calcium concentration and PAD2. CONCLUSIONS: These results suggest calcium modulation could be exploited as therapeutic strategy to modulate pressure-induced PAD2 expression and citrullination.

Activation of protein kinase C reverses capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.

Ca2+ selective ion channels of vanilloid receptor subtype-1 (TRPV1) in capsaicin-sensitive dorsal root ganglion (DRG) neurons and TRPV1 transfected Chinese hamster ovarian (CHO) cells are desensitized following calcium-dependent tachyphylaxis induced by successive applications of 100 nM capsaicin. Tachyphylaxis of TRPV1 to 100 nM capsaicin stimuli was not observed in the absence of extracellular calcium. Capsaicin sensitivity of desensitized TRPV1 ion channels recovered on application of phorbol-12-myristate-13-acetate (PMA). PMA-induced recovery of desensitized TRPV1 was primarily due to influx of extracellular calcium observed during re-application of capsaicin following desensitization. Capsazepine blocked the re-sensitization to capsaicin by PMA. Protein kinase C (PKC) inhibitory peptide PKC fragment 19-36 also inhibited re-sensitization to capsaicin by PMA. Reversal of capsaicin-induced desensitization by PMA was prevented by a mutation of TRPV1 where phosphorylation sites serine502 and serine800 were replaced with alanine. This study provides evidence for a role of PKC in reversing capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.

Membrane topology and membrane retention of the ryanodine receptor calcium release channel.

The ryanodine receptor (RyR) is a Ca2+ release channel located in the sarcoplasmic/endoplasmic reticulum (ER) membrane and plays a critical role in excitation-contraction coupling of skeletal and cardiac muscles. RyR normally exists in a tetrameric structure and contains two functional domains: a carboxyl-terminal hydrophobic domain that contains the conduction pore of the Ca2+ release channel, and a large amino-terminal domain that contains sites responsible for channel regulation. Recent studies involving mutagenesis and heterologous expression have helped unravel the structure-function relationship of RyR, including transmembrane topology and intracellular localization of the Ca2+-release channel. The carboxyl-terminal portion of RyR contains the putative transmembrane segments and is sufficient to form a functional Ca2+-release channel. The amino-terminal region of the protein contains sites responsible for regulation by endogenous modulators such as Ca2+ and Mg2+ and by exogenous ligands such as caffeine. The membrane topology of RyR appears to contain an even number (four or six) of transmembrane segments with a ion selectivity filter present within a region residing between the last two segments, similar to potassium channel, whose atomic structure was described recently. The transmembrane segments also contain sequences that are responsible for localization of RyR in the endoplasmic reticulum, and this sequence is highly conserved in IP3 receptors, which also function as Ca2+-release channels.

Single-channel characterization of the rabbit recombinant RyR2 reveals a novel inactivation property of physiological concentrations of ATP.

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.

Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin.

Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alphaB-crystallin mutants in which R56 was deleted (DeltaR56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (DeltaL55 and DeltaA57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and DeltaR56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in DeltaR56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, DeltaL55 and DeltaA57, showed no significant changes in the chaperone function compared to that of the Wt. The DeltaR56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alphaB-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.

The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.

Ca(2+)-dependent interaction between FKBP12 and calcineurin regulates activity of the Ca(2+) release channel in skeletal muscle.

Calcineurin is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca(2+) dependence. This association involves a Ca(2+) dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca(2+) release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, DeltaCnA(391-521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (DeltaCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca(2+) signaling of muscle contraction and relaxation.

Co-expression of MG29 and ryanodine receptor leads to apoptotic cell death: effect mediated by intracellular Ca2+ release.

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.

A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle.

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.

Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor.

Dantrolene is a drug that suppresses intracellular Ca(2+) release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca(2+) release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [(3)H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [(3)H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro and in vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [(3)H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1-2s in both Western blots and immunoprecipitation assays and specifically inhibits [(3)H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590-609.