Soluble Ligand-binding Domain Research Articles

Effects of Co-Transfection with Myostatin-Targeting siRNA and ActRIIB-Fc Fusion Protein on Skeletal Muscle Growth

Background: Myostatin (Mstn) is a secreted TGF-β family member that controls skeletal muscle growth, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-β family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the combinative effects of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc on murine myoblast in vitro and in vivo. Materials and Methods: C2C12 cells were treated by Mstn-siRNA with or without ActRIIB-Fc at 0 and 48 h after differentiation. Myotube size was measured, and gene expression of Mstn, MuRF-1, MyoD and myogenin were analyzed. Furthermore, 11-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc locally into the masseter muscle twice a week. Histological and biochemical analyses were performed using the dissected muscles. Results: Transfection of Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc resulted in significant increases in the myotube diameter of the C2C12 cells compared with untreated control. Also, treatment with Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc could lead to an upregulation of MyoD and myogenin gene expression and downregulation of Mstn and MuRF-1. In vivo, muscle fibril hypertrophy was observed in both Mstn-siRNA and Mstn-siRNA/ActRIIB-Fc treated groups. Moreover, western blotting analysis showed that the p-Smad2/3 expression level was decreased by treatment of Mstn-siRNA/ActRIIB-Fc. In contrast, MyoD and myogenin protein levels were increased by combined treatment, compared with the other groups. Conclusions: These suggest that double inhibition of myostain is potentially useful for myogenesis and muscle growth promotion. This may be a good as new treatment remedy for patients with various muscle atrophies, including muscular dystrophy.

Co-Administration of Myostatin-Targeting siRNA and ActRIIB-Fc Fusion Protein Increases Masseter Muscle Mass and Fiber Size.

Myostatin, a member of the TGF-β superfamily, is a negative regulator of skeletal muscle cell growth and differentiation, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF-β family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the effectiveness of the co-delivery of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc into skeletal muscle as a potential treatment of atrophic myopathies. Eleven-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA with/without ActRIIB-Fc locally into the masseter muscle twice a week. Inhibition of myostatin function by the combination of Mstn-siRNA and ActRIIB-Fc increased muscle weight and myofibril size in murine masseter muscle. Real-time RT-PCR analysis revealed significant downregulation of myostatin mRNA expression in both the Mstn-siRNA-treated and the combination treatment group. Furthermore, myogenin mRNA expression was upregulated in the combination treatment group, while MuRF-1 and Atrogin-1 mRNA expression was downregulated compared to administration of each compound alone. These findings suggest that double inhibition of myostatin is a potentially useful treatment strategy to increase muscle mass and fiber size and could be a useful treatment of patients with various muscle atrophies, including muscular dystrophy.

X-ray structure of the human α4β2 nicotinic receptor.

Nicotinic acetylcholine receptors are ligand gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and play diverse signaling roles in the central nervous system. The nicotinic receptor has been a model system for cell surface receptors, and specifically for ligand-gated ion channels, for well over a century1,2. In addition to the receptors’ prominent roles in the development of the fields of pharmacology and neurobiology, nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy, and for neuromuscular blocking agents used during surgery2–4. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata5. Structures of a soluble ligand binding domain have provided atomic-scale insights into receptor-ligand interactions6, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism7. All available high-resolution structures are of homopentameric receptors. However, the vast majority of pentameric receptors (called Cys-loop receptors in eukaryotes) present physiologically are heteromeric. Here we present the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. This structure provides insights into the architectural principles governing ligand recognition, heteromer assembly, ion permeation and desensitization in this prototypical receptor class.

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.

On the Mechanisms of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Binding to Glutamate and Kainate

The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediates much of the fast excitatory neurotransmission in the central nervous system. The ability of these receptors to shape such responses appears to be due in part to dynamic processes induced by agonists in the ligand-binding domain. Previous studies employing fluorescence spectroscopy and whole cell recording suggest that agonist binding is followed by sequential transitions to one or more distinct conformational states. Here, we used hydrogen-deuterium exchange to determine the mechanisms of binding of glutamate and kainate (full and partial agonists, respectively) to a soluble ligand-binding domain of GluR2. Our results provide a structural basis for sequential state models of agonist binding and the free energy changes of the associated state-to-state transitions. For glutamate, a multi-equilibrium binding reaction was discerned involving distinct ligand docking, domain isomerization, and lobe-locking steps. In contrast, kainate binding involves a simpler dock-isomerization process in which the isomerization equilibrium is shifted dramatically toward open domain conformations. In light of increasing evidence that the stability, in addition to the extent, of domain closure is a critical component of the channel activation mechanism, the differences in domain opening and closing equilibria detected for glutamate and kainate should be useful structural measures for interpreting the markedly different current responses evoked by these agonists.

Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an "open" conformation, or "lock" the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.

Expression, purification and identification of LBD domain of human PPARδ in E. coli

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ (PPARδLBD) for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted downstream of the malE gene in the vector pMAL-p2x, which encoded maltose-binding protein (MBP), resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E.coli TB1 that was cultured shakily at 30 °C, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.

Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484–509

Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this 'consensus' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.

LRET Investigations of Conformational Changes in the Ligand Binding Domain of a Functional AMPA Receptor

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.

N-Methyl-d-aspartate (NMDA) Receptor Subunit NR1 Forms the Substrate for Oligomeric Assembly of the NMDA Receptor

The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.

A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation

AbstractMembrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of the extracellular domain or alternative mRNA splicing yielding a secreted protein. Mertk (Mer) is in a receptor tyrosine kinase family with Axl and Tyro-3, and all 3 receptors share the Gas6 ligand. Mer regulates macrophage activation, promotes apoptotic cell engulfment, and supports platelet aggregation and clot stability in vivo. We have found that the membrane-bound Mer protein is cleaved in the extracellular domain via a metalloproteinase. The cleavage results in the production of a soluble Mer protein released in a constitutive manner from cultured cells. Significant amounts of the soluble Mer protein were also detected in human plasma, suggesting its physiologic relevance. Cleavage of Mer was enhanced by treatment with LPS and PMA and was specifically inhibited by a tumor necrosis factor α-converting enzyme metalloproteinase inhibitor. As a decoy receptor for Gas6, soluble Mer prevented Gas6-mediated stimulation of membrane-bound Mer. The inhibition of Gas6 activity by soluble Mer led to defective macrophage-mediated engulfment of apoptotic cells. Furthermore, soluble Mer decreased platelet aggregation in vitro and prevented fatal collagen/epinephrine-induced thromboembolism in mice, suggesting a potential therapeutic use for soluble Mer in the treatment of clotting disorders.

Isoform-Specific Early Trafficking of AMPA Receptor Flip and Flop Variants

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-D(flop) receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-D(flip) undergoes complex glycosylation and reaches the plasma membrane at >10x higher levels than GluR-D(flop), as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-D(flop) near to the level exhibited by GluR-D(flip). Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.

Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A

NR3A is expressed widely in the developing CNS of mammals. Coassembly of NR3A with NR1 and NR2 modifies NMDA receptor-mediated responses, reducing calcium permeability and single-channel conductance. The ligand binding properties of NR3A are unknown but shape the role NR3A plays when incorporated into NMDA receptors. Here, a soluble NR3A ligand binding domain (NR3A S1S2) was constructed based on amino acid sequence alignments with other glutamate receptor ion channels and is expressed in Escherichia coli. After purification by affinity, gel filtration, and ion exchange chromatography, NR3A S1S2 behaves as a monomer even at a concentration of 20 mg/ml, as determined by size-exclusion chromatography and dynamic light scattering. NR3A S1S2 has very high affinity for glycine with an apparent dissociation constant (Kd) of 40 nm, 650-fold less than the Kd for NR1. Glutamate, which binds to NR2 subunits, also binds to NR3A, but with very low affinity (Kd = 9.6 mm); in contrast, binding of glutamate to NR1 was not detectable even at a 300 mm concentration. The antagonist binding profiles of NR3A and NR1 also show striking differences. 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), and its analog CGP78608, bind to NR3A S1S2 with low micromolar affinity, whereas for NR1, the affinity of CGP78608 increases 1000-fold compared with CNQX. Other high-affinity NR1 antagonists also show very weak binding to NR3A. Proteolysis protection experiments reveal that CNQX and CGP78608 bind to and stabilize domain 1 of NR3A S1S2 but increase proteolysis of domain 2, indicating that they produce conformational changes distinct from those induced by glycine and D-serine.

The Pro335 → Leu Polymorphism of Type 3 Inositol 1,4,5-Trisphosphate Receptor Found in Mouse Inbred Lines Results in Functional Change

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> Leu polymorphism of mouse IP3R3 between BALB/c and C57BL/6J. We investigated the functional differences between Pro335IP3R3 and Leu335IP3R3 with purified receptors reconstituted into proteoliposomes as well as with soluble ligand binding domains. Pro335IP3R3 exhibited significantly higher IP3-binding affinity and IP3-induced Ca2+ release than those of Leu335IP3R3 in both forms of the receptor. Moreover, the polymorphic change caused differences in the effect of external Ca2+ on IP3-induced Ca2+ release. The Pro335 --> Leu substitution alters the conformation of soluble ligand binding domain as revealed by intrinsic fluorescence and circular dichroism spectra with or without Ca2+. The results indicate that the polymorphism of IP3R3 causes changes in receptor function, presumably affecting intracellular Ca2+ signaling.

LDL receptor family: Isolation, production, and ligand binding analysis

Members of the low density lipoprotein receptor gene family have recently received particular attention because of their involvement not only in lipoprotein transport, but also in signal transduction pathways. The main characteristic feature of this protein group is their cysteine-rich ligand binding domain, which is able to bind many unrelated proteins, such as apolipoproteins, proteases, and protease/inhibitor complexes, signaling molecules such as reelin, and several other groups of proteins. The main challenges of studying these proteins in vitro are their extremely high content of disulfide bridges and the detergent-sensibility of their classical ligands, i.e, lipoproteins. Here, we describe generally applicable procedures for the analysis of these receptors. We present an outline of established methodology for their isolation and visualization, the production of recombinant fragments, in particular of soluble ligand binding domains, and we describe standard procedures for the analysis of the functionality of the receptors and recombinant receptor ligand binding fragments, respectively.

A Soluble Truncated Form of the Mer Tyrosine Kinase Inhibits Gas6 Activity in Platelets and Monocytes.

Several receptor tyrosine kinases generate soluble ligand binding domains either by proteolytic cleavage of the extracellular domain or differential splicing yielding in a truncated RNA transcript. Mer is a member of a receptor tyrosine kinase family which includes Axl and Tyro-3. All three members of this family have an extracellular domain which includes two immunoglobulin and two fibronectin type III motifs. Mer, Axl, and Tyro-3 are transforming in vitro in numerous cell types and are overexpressed in several different human cancers. In addition, Mer, Axl, and Tyro-3 and the common ligand Gas6 are all expressed in platelets and directly affect platelet activation and clot stability. We have found that the 205 kD Mer protein in platelets and monocytes is post-translationally processed by cleavage of the extracellular domain via a TACE-like metalloprotease. The cleavage results in a soluble extracellular domain protein (approximately 150kD) and a membrane-bound kinase domain. The proteolytic cleavage of Mer can be enhanced by LPS and PMA, and specifically inhibited by a TAPI, a TACE inhibitor. Significant amounts of the soluble Mer protein are present in human and mouse serum. The soluble Mer protein directly binds the ligand Gas6, and inhibits Gas6 stimulation of full-length Mer. Gas6, related to anticoagulant protein S, is also a ligand for Axl and Tyro-3. The cleavage of Mer may represent a mechanism of directly regulating the numerous functions of Gas6 including promoting platelet adhesion and clot stability, stimulating cell proliferation, inducing cell adhesion and chemotaxis, and preventing apoptosis. Furthermore, the ability of soluble Mer to inhibit Gas6 activity in platelets may provide a potential novel therapeutic intervention in the treatment of clotting disorders.

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.

Determinants of antagonist binding at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit, GluR-D. Role of the conserved arginine 507 and glutamate 727 residues.

Previous structural and mutagenesis studies indicate that the invariant alpha-amino and alpha-carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR-D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand-binding properties of homomeric GluR-D and its soluble ligand-binding domain with mutations at these positions. Filter-binding assays using [3H]AMPA, an agonist, and [3H]Ro 48-8587, a high-affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high-affinity binding for [3H]Ro 48-8587, displaceable by 7,8-dinitroquinoxaline-2,3-dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist-specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.

Synthesis and Purification of Soluble Ligand Binding Domain of the Human Vitamin D3Receptor

We expressed and purified milligram quantities of the ligand binding domain of the human 1,25-dihydroxyvitamin D3receptor using a glutathione-S-transferase (GST) fusion protein expression system. Amino acids 105–427 were expressed inE. colias a GST fusion protein at a reduced (20°C) temperature and purified on glutathione sepharose. The fusion protein adsorbed to glutathione sepharose was cleaved with thrombin to yield soluble 105–427 human 1,25-dihydroxyvitamin D3receptor. The 105–427 human 1,25-dihydroxyvitamin D3receptor was further purified by Mono Q ion exchange chromatography and was characterized as a single band on SDS–polyacrylamide gel electrophoresis. The 105–427 human 1,25-dihydroxyvitamin D3receptor bound 1,25-dihydroxyvitamin D3with high affinity (Kdapproximately 10−9M) and with a binding capacity of 47 pmoles/nmole protein. Large scale expression of 105–427 human 1,25-dihydroxyvitamin D3receptor will provide human 1,25-dihydroxyvitamin D3receptor ligand binding domain suitable for structural studies.