Substrate For Horseradish Peroxidase Research Articles

A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin‐film biosensor chips

Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.

Regulation of Gephyrin Assembly and Glycine Receptor Synaptic Stability

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting

O-269: Development of a sensitive assay to measure lactate production of preimplantation embryos

Reliable indicators of embryo viability are rate of glucose/pyruvate consumption and lactate production. The ability to non-invasively quantify embryo metabolism within culture media is a valuable tool. Current non-invasive methods of measuring embryo metabolism are based on generation or utilization of reduced pyridine nucleotides, NADH and NADPH. This method requires excitation of NADH/NADPH with ultraviolet (UV) light which can damage embryo DNA thus requiring manual removal of media, nanoliter pipetting, and dilutions that can introduce quantitative errors. Long-term goals of these studies are to develop metabolic assays that can be performed in microfluidic embryo culture and analysis platforms that remove manual media manipulation and dilution, do not use UV light, and are “embryo-safe”. Study objectives were to optimize a system of measuring single embryo-produced lactate that would be compatible with microfluidic culture without use of UV light detection. Experimental study. Lactate assays were developed using Amplex UltraRed (AR), which is a colorless, stable and versatile fluorogenic substrate for horseradish peroxidase (HRP). In this assay, lactate oxidase (LOX) reacts with L-lactate to form pyruvate and H2O2. In the presence of HRP, H2O2 reacts with AR reagent in a 1:1 stoichiometry to generate a red-fluorescent oxidation product, resorfin. Amplex UltraRed lactate assays were optimized with 100 μM AR reagent, 0.2 U/ml HRP and 2U/ml LOX in three different reaction buffers (1X reaction buffer, glycine-hydrazine buffer, and 1X reaction buffer containing glycine and hydrazine) at pH 6.5, 7.5, and 8.5 over a span of 0-3 h. Standard curves of L-lactate in KSOM were established using 10-fold serial dilutions from 10nM to10mM. Lactate was measured in presence or absence of pyruvate and glucose to rule out interference from other substrate sources. A 3X3 cross-over design was used to compare assay sensitivity under varying concentrations of AR reagent (100, 10, and 1 μM) and HRP (0.2, 0.02, and 0.002 U/ml). Lactate was quantified with an excitation filter at 535 nm and an emission filter at 580 nm. Two-cell embryos from B6SJL-F2 mice were individually cultured in 10 μl KSOM w/o L-lactate for 3 h at 37°C in 5% CO2 in air. Embryo culture media was assayed at a 1:1 ratio with AR. Triplicate standard curves measuring 10-fold dilutions of lactate were performed each time embryos were assessed for lactate. The optimum buffer conditions for AR lactate assays was 1X reaction buffer at pH 7.5. There was no effect of glucose or pyruvate on AR lactate assay sensitivity. Reducing AR reagent to 10 μM and HRP to 0.002 U/ml greatly reduced background fluorescence and decreased the limit of detection to 600 nM lactate. Individual 2-cell embryo lactate production was measured at 44.9 ± 4.4 pmol/ 3h. Amplex UltraRed is a sensitive reagent that can be used to measure the production of lactate in the picomole range. These results provide a new method to non-invasively quantify preimplantation embryo metabolism in the absence of a UV light source. Future studies will focus on applying this technology to a microfluidic embryo culture and analysis system for real-time monitoring of embryo metabolism.

Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents

INTRODUCTIONEnzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction products are slate gray to black, and they are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains.

Flow-Through Multianalyte Chemiluminescent Immunosensing System with Designed Substrate Zone-Resolved Technique for Sequential Detection of Tumor Markers

A novel flow-through immunosensing system for performing a multianalyte chemiluminescent determination in a single run was designed. A new analytical strategy of substrate zone-resolved technique was proposed. Using carcinoma antigen 125 (CA 125) and carcinoembryonic antigen (CEA) as model analytes, the capture antibodies for CA 125 and CEA were immobilized on an UltraBind aldehyde-activated membrane to act as an immunoreactor, to which the mixture of CA 125, CEA, and their corresponding tracers, horseradish peroxidase (HRP)-labeled anti-CA 125 and alkaline phosphatase (ALP)-labeled anti-CEA, was introduced for on-line incubation. The substrates for HRP and ALP were then delivered into the detection cell sequentially to perform substrate zone-resolved immunoassay by a sandwich format. Under optimal conditions, CA 125 and CEA could be assayed in the ranges of 5.0-100 units/mL and 1.0-120 ng/mL, respectively. The whole assay process including incubation, wash, detection, and regeneration could be completed in 35 min. The serum samples from the clinic were assayed with the proposed method, and the results were in acceptable agreement with the reference values. This method and the strategy of substrate zone-resolved technique could be further developed for high-throughput multianalyte immunoassay.

Development of a Chemiluminescent ELISA for Determining Chloramphenicol in Chicken Muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.

Electrochemical Enzyme‐Linked Immunoassay for the Determination of Estriol Using Methyl Red as Substrate

A new electrochemical substrate for horseradish peroxidase, methyl red, is reported. In this reaction system, horseradish peroxidase can catalyze the redox reaction of methyl red and H2O2. Methyl red exhibits a sensitive voltammetric peak at−0.51 V vs. Ag/AgCl reference electrode, the decrease of the peak current of methyl red is in proportion to the concentration of horseradish peroxidase (HRP). The linear range for determination of horseradish peroxidase is 5.0×10−8∼5.0×10−7 g mL−1 and the detection limit is 1.8×10−8 g mL−1. The relative standard deviation is 3.3% when 2.0×10−7 g mL−1 HRP was sequentially determined 11 times. A voltammetric enzyme‐linked immunoassay method for the determination of estriol was developed, based on this electrochemical system. The linear range for determination of estriol is 1.0∼1000.0 ng mL−1, and the detection limit is 0.33 ng mL−1. The relative standard deviation for 11 parallel determinations with 200 ng mL−1 estriol is 4.8%. Some pregnancy serum samples were analyzed with satisfactory results.

CD163 Identifies Perivascular Macrophages in Normal and Viral Encephalitic Brains and Potential Precursors to Perivascular Macrophages in Blood

Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.

Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.

15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

Characterization of the Gαs Regulator Cysteine String Protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Iron(III)protoporphyrin/MCM41 catalyst as a peroxidase enzyme model: Preparation and typical test reactions

MCM41 was synthesized and Fe(III)protoporphyrin(IX) (Hemin, Fe(III)PPIX) was directly encapsulated into MCM41 pores through the self-assembly formation mechanism using micelles of quaternary alkylammonium salt (cetyl trimethyl ammonium bromide) as the solvent for Fe(III)PPIX and template for formation of MCM41 spontaneously. The catalysts obtained were characterized by XRD, BET N 2 adsorption isotherm, diffuse reflectance UV–vis and FT-IR techniques. Fe(III)PPIX/MCM41 was used in some typical test reactions, such as oxidation of phenol, ortho-methoxyphenol (guaiacol) and synthesis of indophenol. Kinetic parameters including initial reaction rates, rate constants, V max, turnover number, Michaelis constant and catalytic efficiency were obtained and compared to those of Fe(III)/MCM41(as a blank) and horseradish peroxidase (HRP) enzyme (consisting of Fe(III)PPIX as the prosthetic group). Results showed that Fe(III)PPIX/MCM41 catalyst was able to mimic horseradish peroxidase with a K m value of 183 μM with respect to guaiacol (as the reducing substrate of HRP). Fe(III)PPIX/MCM41 catalyzes the oxidation reactions (in the presence of hydrogen peroxide) at higher rates than the homogeneous Fe(III)PPIX system and Fe(III)/MCM41 heterogeneous catalyst, but at lower rates than the enzyme as a homogeneous catalytic system.

Chemiluminescence immunoassay for chloramphenicol

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87-100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1-10 ng/ml, and it displayed good linearity.

Tpl2/Cot Signals Activate ERK, JNK, and NF-κB in a Cell-type and Stimulus-specific Manner

Macrophages and B-cells from Tpl2 knock-out mice exhibit a restricted defect in lipopolysaccharide and death receptor signaling that is limited to the activation of ERK. Here we show that Tpl2-/- MEFs exhibit defects in ERK, JNK, and NF-kappaB activation, or ERK activation only when stimulated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, respectively. In addition, we show that the activation of Tpl2 by TNF-alpha depends on signals transduced by both TRAF2 and RIP1. Activated Tpl2 phosphorylates MKK4/SEK1 upstream of JNK and stimulates NF-kappaB DNA binding and transcriptional activity by mechanisms that are independent of the nuclear translocation of p50 and p65. Tpl2-transduced TNF-alpha signals instead promote the phosphorylation of p65 at Ser276 and modulate the spectrum of proteins associated with p65. Phosphorylation stimulates the transcriptional activity of NF-kappaB but does not affect its ability to bind DNA, which may be affected by the composition of the nuclear NF-kappaB complexes. These data confirm that defects caused by a single mutation may be cell-type and signal-specific and delineate the role of Tpl2 in the transduction of TNF-alpha signals that activate JNK and NF-kappaB in MEFs.

Gold-film array-electrode for electrochemical ELISA

A gold-film array-electrode (8-channel) has been developed for application to an electrochemical ELISA system. The principle of the electrochemical ELISA is the amperometric quantification of oxidized 3,5,3′,5′-tetramethylbenzidine (TMB) which is widely used as a chromogenic substrate of horseradish peroxidase (HRP) in commercial ELISA kits. To make an 8-channel array-electrode, eight sets of three-electrode systems were integrated with the spacing of a 96-well microplate. The gold-film electrode for working- and auxiliary-electrode was made by sputtering a gold layer on a polyetherimide (PEI) strip, and the reference electrode was made by spin coating with Ag/AgCl paste. Using the gold-film electrode, the measurement parameters, such as analysis time, electrode area, measuring temperature and so on, were optimized. Using 50 array-electrodes, the electrode-to-electrode difference in sensitivity was estimated to be 8.2%. To demonstrate the practical applicability of the gold-film electrode, it was applied to commercial ELISA kits for the detection of HIV (antibody), HBV (antigen and antibody), and HCV (antibody). The life time of the gold-film electrode was estimated for use for over 500 repetitive measurements.

Transition Metal Chemistry of Glucose Oxidase, Horseradish Peroxidase, and Related Enzymes

AbstractFor Abstract see ChemInform Abstract in Full Text.

Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

Dual Function for a Unique Site within the β2I Domain of Integrin αMβ2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.

Ligand Binding Is a Critical Requirement for Plasma Membrane Expression of Heteromeric Kainate Receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.