Intracellular C-terminal Domain Research Articles

Molecular Basis of Tetramerization and pH-Gating in the KcsA Potassium Channel Cytoplasmic Domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue long segment which natively adopts a helical bundle conformation with four-fold symmetry. A hallmark of KcsA behavior is a pH-induced conformational change which leads to opening of the channel at acidic pH. While crystal structures of full-length KcsA failed to observe a pH-effect upon the CTD, other biophysical methods have presented evidence to the contrary. We approached the question of CTD structure and its pH-dependence by studying the behavior of soluble peptides corresponding to residues 128-160 of the CTD (CTD34). A combination of NMR and sedimentation equilibrium experiments established CTD34 to be a tetramer with a KD of (2.0 ± 0.5) x 10−11 M3 at neutral pH, and this tetrameric species undergoes pH-dependent dissociation, rendering CTD34 fully monomeric below pH 5.0. The structural basis for this phenomenon is destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide.Molecular factors contributing to CTD tetramerization were investigated by comparing the tetrameric stability of single alanine mutants as determined by NMR, SE and molecular dynamics. Single-residue contributions to tetramer stability were in the 0.5-3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than the inter-subunit salt-bridges between R147 and D149/E152. A third class of residues outside the helical interface influenced tetramer stability via the tetramerization on-rate by changing the inherent helical propensity of CTD34 which promotes tetramer formation. We conclude that the CTD is an independent tetramerization domain modulated by pH, and tetramer formation is controlled by a combination of on- and off-rate effects which conform to current paradigms of protein folding.

Effects of External and Internal Ca2+ on Unitary NMDA Receptor Properties

N-Methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels widely expressed throughout the central nervous system. When active, they generate electric and biochemical signals by fluxing Na+ and Ca2+ into the post-synaptic cell. The NMDA receptor-mediated Ca2+ transient initiates internal cascades that result in synaptic plasticity and excitotoxicity. In turn, Ca2+ regulates channel activity directly and indirectly through allosteric and 2nd messenger mechanisms that are poorly understood. We investigated the effects of external and internal Ca2+ concentrations on recombinant GluN1/GluN2A (N1/2A) receptors using single-channel current recordings, statistical analysis, and kinetic modeling. Increasing concentrations of extracellular Ca2+ reduced the unitary channel conductance from 75.9±1.4 pS in 0 mM [Ca2+]e, to 55.2±1.6 pS in 1.8 mM [Ca2+]e, and to 11.8±0.7 pS in 75 mM [Ca2+]e. Importantly, receptors lacking the intracellular C-terminal domain (CTD) of the N1 subunit (N1Δ/2A) but not the 2A subunit (N1/2AΔ) exhibited higher unitary conductance in the absence of external Ca2+ (N1Δ/2A γ = 83.3±1.0 pS, N1/2AΔ γ = 79.2±1.4 pS). This is consistent with a role for the N1 CTD in setting channel conductance for Na+. However, in the presence of physiological external Ca2+ (1.8 mM), both exhibited N1Δ/2A (γ = 50.4±1.2 pS, p<0.05) and N1/2AΔ (γ = 60.8±2.0 pS, p<0.05) channels had a lower conductance relative to wild-type. Based on these novel results, we asked whether this Ca2+-dependent regulation of conductance depends on calmodulin binding to either the C0 or C1 cassettes of N1. Furthermore, we asked whether local Ca2+ influx through the NMDA receptor pore is sufficient for this regulation. Our results help to further unravel the Ca2+-dependent processes that control the properties of individual NMDA receptor channels.

Amyloid Beta Precursor Protein: Proper Credit for the Basic Biochemical Properties of the Most Studied Protein in the 21st Century*

Introduction The amyloid precursor protein (APP) is mainly known for being the precursor of the s-amyloid peptide, which accumulates in plaques found in the brain of Alzheimer’s disease patients. Expression in different tissues and the degree of sequence identity among mammals indicate an essential and non-tissue specific physiological function. APP is anchored to the membrane and displays a single C-terminal intracellular domain and a longer N-terminal extracellular domain. The basic biochemical properties and the scattered data on research, not related to production of beta-amyloid peptide, suggest that the protein and the molecules resulting from APP proteolytic cleavage may act as adhesion factors, enzymes, hormones/neurotransmitters and/or protease inhibitors. APP deserves to be known for its quite notable properties and its physiological role(s). Citation: Franco R, Navarro G, Martinez-Pinilla E, Moreno E (2014) Amyloid Beta Precursor Protein: Proper Credit for the Basic Biochemical Properties of the Most Studied Protein in the 21st Century. J Neurol Neurol Disord 1(1): 103 The number of papers related to amyloid precursor protein (APP) as of September 2014 is 30,075. Of those, papers in this Century are 24,390 (81% of the total), indicating that APP is one of the most studied proteins. Information is useful but excess of data may be counterproductive by the risk of losing the perspective to fully apprehend APP biological clues. A key issue in understanding APP biological relevance is to focus on its physiological role(s). As every other protein, APP may have different roles; a main one and other(s) possibly less relevant. This certainty drives scientific research. Due to enormous interest related to accumulation of a degradation peptide, beta-amyloid (As) peptide, in the brain of Alzheimer’s disease (AD) patients, APP research is biased towards the central nervous system (CNS) and mainly driven by its relationship with the amyloid-hypothesis of AD. The protein is widely expressed and is highly conserved among species; therefore its physiological function must be essential and non-tissue specific, i.e. not necessarily centered on the nervous system. In this perspective paper the focus is placed on aspects apparently attracting less interest in the field, namely the possibility of i) a relevant role of carbohydrates in APP function, ii) differential APP glycosylation in AD, iii) APP acting as adhesion molecule, iv) APP having enzyme activity, v) APP proteolytic cleavage providing polypeptides with hormone/neurotransmitter/growth factor potential, and vi) soluble APP acting as protease inhibitor in the CNS.

The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn2+ binding to this domain

The voltage-gated proton channel Hv1 is strongly sensitive to Zn2+. The H+ conduction is decreased at a high concentration of Zn2+ and Hv1 channel closing is slowed by the internal application of Zn2+. Although the recent studies demonstrated that Zn2+ interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn2+ binds to His244 and His266 residues. The thermodynamics signature of Zn2+ binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn2+ to His244 (mutant H266A) and His266 (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

The DRY box and C-terminal domain of the human cytomegalovirus US27 gene product play a role in promoting cell growth and survival.

Human cytomegalovirus (HCMV) is a widespread pathogen that can lay dormant in healthy individuals and establish lifelong latent infection. This successful co-existence is facilitated by a number of viral gene products that manipulate host cellular functions and immune responses. Among these immunomodulatory genes are four G-protein coupled receptors (GPCRs) encoded by HCMV, designated US27, US28, UL33, and UL78. Studies have shown the US28 gene product to be a functional chemokine receptor that signals both constitutively and in a ligand-dependent manner, resulting in a wide range of cellular effects. In previous work, we have found that US27 expression results in at least two biological effects: enhanced CXCR4 signaling and increased in cellular proliferation in HEK293 cells. Here, we examined the involvement of two protein domains, the DRY box and the C-terminal intracellular domain (CTD) of US27, in mediating both cell proliferation and survival. While both domains were required for a proliferative effect, loss of either domain only moderately impacted cell survival, suggesting that US27 may interact with cell survival pathways through protein regions other than the DRY box and CTD. Quantitative RT-PCR arrays were used to profile changes in cellular gene expression in the HEK293-US27 cell line, and down-regulation of cell cycle regulators CDKN1A/p21/CIP1 (cyclin dependent kinase inhibitor 1A) and SESN (Sestrin2 or Hi95) was observed. These results indicate that increased cell proliferation due to US27 may be linked to suppression of negative growth regulators, and that these interactions require the DRY box and CTD.

The planar cell polarity protein Vangl2 bidirectionally regulates dendritic branching in cultured hippocampal neurons.

BackgroundVan Gogh-like (Vangl) 2 is a planar cell polarity (PCP) protein that regulates the induction of polarized cellular and tissue morphology during animal development. In the nervous system, the core PCP signaling proteins have been identified to regulate neuronal maturation. In axonal growth cones, the antagonistic interaction of PCP components makes the tips of filopodia sensitive to guidance cues. However, the molecular mechanism by which the PCP signaling regulates spine and dendritic development remains obscure.FindingsHere we explored the finding that a loss of function of Vangl2 results in a significant reduction in spine density and complexity of dendritic branching. In spite of a previous report, in which the Vangl2 C-terminal TSV motif was shown to be required for the interaction with PSD-95 and the C-terminal intracellular domain was shown to associate with N-cadherin, overexpression of deletion mutants (Vangl2-∆TSV and Vangl2-∆C) had little effect on spine density. However, when an N-terminal region deletion mutant was overexpressed, spine density was slightly down-regulated. Intriguingly, the deletion mutants had a more potent effect on dendritic branching, such that the deletion of the N-terminal region reduced dendritic branching, whereas deletion of the C-terminal region increased it.ConclusionsBased on these results, Vangl2, a core PCP signaling pathway component, appears to have a functional role in neural complex formation. Especially in the case of dendritic branching, Vangl2 serves as a molecular hub to regulate neural morphology in opposite directions.

Molecular and genetic determinants of the NMDA receptor for superior learning and memory functions.

The opening-duration of the NMDA receptors implements Hebb's synaptic coincidence-detection and is long thought to be the rate-limiting factor underlying superior memory. Here, we investigate the molecular and genetic determinants of the NMDA receptors by testing the “synaptic coincidence-detection time-duration” hypothesis vs. “GluN2B intracellular signaling domain” hypothesis. Accordingly, we generated a series of GluN2A, GluN2B, and GluN2D chimeric subunit transgenic mice in which C-terminal intracellular domains were systematically swapped and overexpressed in the forebrain excitatory neurons. The data presented in the present study supports the second hypothesis, the “GluN2B intracellular signaling domain” hypothesis. Surprisingly, we found that the voltage-gated channel opening-durations through either GluN2A or GluN2B are sufficient and their temporal differences are marginal. In contrast, the C-terminal intracellular domain of the GluN2B subunit is necessary and sufficient for superior performances in long-term novel object recognition and cued fear memories and superior flexibility in fear extinction. Intriguingly, memory enhancement correlates with enhanced long-term potentiation in the 10–100 Hz range while requiring intact long-term depression capacity at the 1–5 Hz range.

DYRK1A-mediated phosphorylation of GluN2A at Ser(1048) regulates the surface expression and channel activity of GluN1/GluN2A receptors.

N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

Dimerization of the EphA1 receptor tyrosine kinase transmembrane domain: Insights into the mechanism of receptor activation.

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape.

Apoptosis in Alzheimer's disease: an understanding of the physiology, pathology and therapeutic avenues.

Alzheimer's disease (AD) is a devastative neurodegenerative disorder with complex etiology. Apoptosis, a biological process that plays an essential role in normal physiology to oust a few cells and contribute to the normal growth, when impaired or influenced by various factors such as Bcl2, Bax, caspases, amyloid beta, tumor necrosis factor-α, amyloid precursor protein intracellular C-terminal domain, reactive oxygen species, perturbation of enzymes leads to deleterious neurodegenerative disorders like AD. There are diverse pathways that provoke manifold events in mitochondria and endoplasmic reticulum (ER) to execute the process of cell death. This review summarizes the crucial apoptotic mechanisms occurring in both mitochondria and ER. It gives substantial summary of the diverse mechanisms studied in vivo and in vitro. A brief account on neuroprotection of several bioactive components, flavonoids and antioxidants of plants against apoptotic events of both mitochondria and ER in both in vitro and in vivo has been discussed. In light of this, the burgeoning need to develop animal models to study the efficacy of various therapeutic effects has been accentuated.

Structural Basis of a Kv7.1 Potassium Channel Gating Module: Studies of the Intracellular C-Terminal Domain in Complex with Calmodulin

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

Gating Characteristics Control Glutamate Receptor Distribution and Trafficking In Vivo

Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking invivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.

Molecular cloning, characterization, and expression analysis of luteinizing hormone receptor gene in turbot (Scophthalmus maximus).

The luteinizing hormone receptor (LHR) plays a crucial role in female reproduction. In the present study, full-length sequence coding for the LHR was obtained from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. The full-length LHR cDNA was 3,184 bp long and contained a 2,058-bp open reading frame which encoded a protein of 685 amino acids. Multiple sequence alignments of the turbot LHR manifested high homologies with the corresponding sequences of available teleosts and representative vertebrates, and significant homology with that of Hippoglossus hippoglossus. In addition, the turbot LHR showed typical characteristics of glycoprotein receptors, including a long N-terminal extracellular domain, seven transmembrane domains, and a short C-terminal intracellular domain. LHR mRNA was abundant in the ovary, but was deficient in extra-ovarian tissues. Furthermore, LHR mRNA gradually developed from previtellogenesis to migratory nucleus stage, with the highest values observed in migratory nucleus stage during reproductive cycle. However, LHR mRNA sharply decreased in atresia stage. These results suggested that LHR is a typical G protein-coupled receptor that is involved in the promotion of turbot ovarian development and may be related to the final maturation and ovulation of oocyte. These findings contribute to the understanding of the potential roles of LHR in controlling the fish reproductive cycle.

Glycogen Synthase Kinase-3β Stabilizes the Interleukin (IL)-22 Receptor from Proteasomal Degradation in Murine Lung Epithelia

Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection as part of type 17 immunity. Molecular regulation of IL-22 receptor (IL-22R) protein levels is unknown. In murine lung epithelia, IL-22R is a relatively short-lived protein (t½ ∼1.5 h) degraded by the ubiquitin proteasome under normal unstimulated conditions, but its degradation is accelerated by IL-22 treatment. Lys(449) within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site as disruption of this site by deletion or site-directed mutagenesis creates an IL-22R variant that, when expressed in cells, is degradation-resistant and not ubiquitinated. Glycogen synthase kinase (GSK)-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser(410) and Ser(414), and IL-22 treatment of cells triggers GSK-3β inactivation. GSK-3β overexpression results in accumulation of IL-22R protein, whereas GSK-3β depletion in cells reduces levels of the receptor. Mutagenesis of IL-22R at Ser(410) and Ser(414) results in receptor variants that display reduced phosphorylation levels and are more labile as compared with wild-type IL-22R when expressed in cells. Further, the cytoskeletal protein cortactin, which is important for epithelial spreading and barrier formation, is phosphorylated and activated at the epithelial cell leading edge after treatment with IL-22, but this effect is reduced after GSK-3β knockdown. These findings reveal the ability of GSK-3β to modulate IL-22R protein stability that might have significant implications for cytoprotective functions and therapeutic targeting of the IL-22 signaling axis.

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1.

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His(244) or His(266) residue. The binding of Zn(2+) to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co(2+), Ca(2+), Ni(2+), Mg(2+), and Mn(2+). The strength of Ca(2+), Co(2+), Mg(2+), Mn(2+) and Ni(2+) binding to the site close to His(244) is stronger than that of these divalent metal ions binding to the site close to His(266).

Interleukin 22 receptor abundance and signaling is regulated by the ubiquitin proteasome and glycogen synthase kinase 3β. (INM2P.422)

Abstract Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection at mucosal and other epithelial surfaces. Molecular regulation of IL-22 receptor (IL-22R) protein has not been characterized. In mouse lung epithelia, IL-22R is expressed as a relatively short-lived protein and is degraded by the ubiquitin proteasome. Its degradation is accelerated when cells are stimulated by IL-22. Lys 449 within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site for proteasomal degradation as deletion or mutation of K449 creates a degradation resistant IL-22R mutant protein that is not ubiquitinated in cells. IL-22 treatment of cells triggers glycogen synthase kinase (GSK)-3β inactivation, and GSK-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser 410. GSK-3β overexpression results in accumulation of IL-22R protein while GSK-3β depletion in cells reduces levels of the receptor, and this effect is abolished on mutation of the 410-414 phosphorylation domain. Signal transduction of IL-22 appears increased when GSK-3β is overexpressed. This study shows GSK-3β as a modulator of IL-22R protein stability, which may be important for host defense and could impact therapeutic targeting of the IL-22 signaling axis.

MOLECULAR EVOLUTION OF GPCRS: Kisspeptin/kisspeptin receptors

Following the discovery of kisspeptin (Kiss) and its receptor (GPR54 or KissR) in mammals, phylogenetic studies revealed up to three Kiss and four KissR paralogous genes in other vertebrates. The multiplicity of Kiss and KissR types in vertebrates probably originated from the two rounds of whole-genome duplication (1R and 2R) that occurred in early vertebrates. This review examines compelling recent advances on molecular diversity and phylogenetic evolution of vertebrate Kiss and KissR. It also addresses, from an evolutionary point of view, the issues of the structure-activity relationships and interaction of Kiss with KissR and of their signaling pathways. Independent gene losses, during vertebrate evolution, have shaped the repertoire of Kiss and KissR in the extant vertebrate species. In particular, there is no conserved combination of a given Kiss type with a KissR type, across vertebrate evolution. The striking conservation of the biologically active ten-amino-acid C-terminal sequence of all vertebrate kisspeptins, probably allowed this evolutionary flexibility of Kiss/KissR pairs. KissR mutations, responsible for hypogonadotropic hypogonadism in humans, mostly occurred at highly conserved amino acid positions among vertebrate KissR. This further highlights the key role of these amino acids in KissR function. In contrast, less conserved KissR regions, notably in the intracellular C-terminal domain, may account for differential intracellular signaling pathways between vertebrate KissR. Cross talk between evolutionary and biomedical studies should contribute to further understanding of the Kiss/KissR structure-activity relationships and biological functions.

Use of Bioluminescence Resonance Energy Transfer (Bret) for Studies of Protein-Protein Interactions Between the GLUD2 Receptor and Intracellular Interaction Partners

The GluD2 receptor is a member of the ionotropic glutamate receptor (iGluR) superfamily and is expressed in cerebellar Purkinje cells (PCs). GluD2 receptors recognize the neurotransmitters D-serine, but lack excitatory ligand-gated ion channel activity. Recently, D-serine activity mediated via GluD2 has been shown to modulate cerebellar long-term-depression (LTD). The intracellular C-terminal domain (CTD) of GluD2 is required for D-serine regulation. A working hypothesis for this regulation is that D-serine binding to the extracellular ligand-binding domain is translated to intracellular rearrangement of the CTD and hereby potentially affects protein-protein interactions (PPIs) with intracellular scaffolding proteins or signaling enzymes involved in expression of cerebellar LTD. Various interacting partners of the GluD2 CTD have been identified; including the PDZ-domain containing scaffolding protein S-SCAM and Delphilin and the tyrosine kinase PTPMEG. Furthermore, the CTD contains multiple phosphorylation sites, which may regulate PPIs.In the present study, our aim is to study the potential regulation of GluD2 PPIs by phosphorylation and D-serine. For this purpose, we have established a bioluminescence resonance energy transfer (BRET) assay that enables measurement of GluD2 PPIs in intact cells by inserting the resonance energy donor Renilla luciferase 8 (Rluc8) at various positions in full-length GluD2 or the isolated CTD and acceptor molecule green fluorescence protein 2 (GFP2) to known GluD2 interaction partners.For PSD95, Delphilin, AP-4 and S-SCAM, it was possible to detect robust BRET between GluD2 and GFP2-tagged full-length interacting partners as well as truncated versions containing the isolated PDZ domain. Hereby, an experimental platform has been created for studying GluD2 PPIs in intact cells. Results from ongoing characterizations of the influence of CTD phosphorylation and D-serine ligand-binding on PPIs will be presented.

Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.