Characterization of monoclonal antibodies directed against the rat neurotensin receptor NTS1

G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a variety of ligands and represent major drug targets. Despite their medical importance, detailed structural information is limited because only one GPCR has been crystallized and its structure determined. To develop tools to aid in the formation of well-ordered crystals, we generated monoclonal antibodies with high affinity to the rat neurotensin receptor. All antibodies bound to the C-terminus of the receptor, which may reflect the selection strategy used to identify high-affinity binders. Further characterization revealed that some antibodies bound to the receptor in a sodium chloride sensitive manner, but others did not. Epitope mapping revealed distinct antigenic regions within the receptor C-terminus. Tight binding of Fab fragments to the receptor was verified by size exclusion chromatography.     

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons.     

An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies

Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.     

Antipeptide Polyclonal Antibodies that Recognize a Substance P-Binding Site in Mammalian Tissues: A Biochemical and Immunocytochemical Study

Abstract: We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using ¹²⁵I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.     

Characterization of Pharmacologically Active Anti-Peptide Antibodies Directed Against the First and Second Extracellular Loops of the Serotonin 5-HT1A Receptor

Abstract: The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT_1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96–111), Ab-2 (loop II; sequence G-W-R-T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173–193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT_1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT_1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT_1A receptor (K_DH = 0.54 ± 0.09 nM and K_DL = 13.74 ± 4.9 nM) for the agonist 8-hydroxy-2-(di-n-[³H]propylamino)tetralin ([³H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (K_D of ≈2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the B_max of the [³H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.     

C-terminal antibodies (CTAbs): a simple and broadly applicable approach for the rapid generation of protein-specific antibodies with predefined specificity

Recent advances in proteomic techniques have resulted in an ever-increasing need to produce antibodies. Here, to address this problem, a technically simple approach of targeting the extreme C-termini of proteins with antibodies (CTAbs) was investigated in proteins secreted by the human pathogen Streptococcus pyogenes. Target proteins were identified by a conventional proteomic approach and CTAbs produced against synthetic five amino acid peptides representing the C-terminus of each target protein. In every case where protein secretion was demonstrated (n = 20), CTAbs were successfully produced and bound specifically to the target protein (100% success rate). The apparent specificity was consistent with the structural heterogeneity of the C-termini of S. pyogenes proteins. The global specificity of CTAb binding was defined using a combinatorial library of synthetic peptides representing structural variants of the original synthetic immunogen. This is a systematic and comprehensive approach for the development of antibodies with defined specificity that can be used in a range of applications.     

Two Distinct Conformations of Aβ Aggregates on the Surface of Living PC12 Cells

Aβ42 has been found to associate rapidly to neuronal cells and is the primary constituent of senile plaques. In this study we monitored the aggregation of Aβ42 with living PC12 cells. Using photobleaching Förster resonance energy transfer, we observed one set of aggregates that displayed colocalization and another that displayed energy transfer. Cell surface aggregates were found to become resistant to potassium iodide (KI)-induced quenching. Exposed Aβ42 regions were probed with three monoclonal antibodies directed against the N-terminus, an internal sequence, and the C-terminus of Aβ42. Two populations of aggregates were revealed: one that bound all three antibodies, and one that bound all but the C-terminus antibody. Of interest, using fluorescent recovery after photobleaching, we observed no Aβ42 exchange within either type of aggregate. These findings offer what we believe is new insight into the conformations of Aβ42 that accumulate on the surface of living cells. One conformation is incapable of energy transfer, is sensitive to KI, and binds C-terminus-specific antibodies. The other conformation increases in number over time, is capable of energy transfer, is quencher-resistant, and has a sequestered C-terminus. With further studies to characterize Aβ aggregation on live cells, the underlying mechanisms leading to Alzheimer's disease may be revealed.     

Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts

Luteinizing hormone and gonadotropin releasing hormone receptors (LHR and GnRHR, respectively) are G protein-coupled receptors with important functions in reproduction. We have developed chimeric GnRHR-LHR that contain the full GnRHR coupled to various forms of the LH receptor C-terminus to explore the role of the LH receptor C-terminus in raft localization of the receptor and signaling. Addition of the full-length LHR C-terminus to GnRHR resulted in localization of the resting chimeric receptor in the bulk membrane rather than plasma membrane rafts as has been reported for the wild-type GnRHR [A. Navratil, S. Bliss, K. Berghorn, J. Haughian, T. Farmerie, J. Graham, C. Clay, M. Roberson, Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK, J. Biol. Chem. 278 (2003) 31593-31602]. With truncation of the LHR C-terminus, approximately 3% of chimeric receptors appeared in low density membrane fractions. Palmitoylation of sites on the LHR C-terminus appears important for raft localization. Mutations to C-terminus palmitoylation sites eliminated translocation of LH receptors from the bulk membrane to rafts upon binding of hCG although these mutant receptors retained the ability to signal via cAMP.     

Identification of a highly specific and versatile vitamin D receptor antibody

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is critical for regulation of serum calcium and phosphorus levels and for proper maintenance of bone mineralization and neuromuscular function. Biological effects of 1,25(OH)₂D₃ are mediated through a nuclear steroid hormone receptor, known as the vitamin D receptor (VDR). The discovery of VDR in a number of different cell and tissue types, suggests that the physiological role of vitamin D may extend beyond the regulation of calcium homeostasis and bone function. Unfortunately, identification of tissues expressing VDR has been controversial due to low abundance of the receptor and quality of the antibodies used. Therefore, we elected to characterize a panel of commercially available VDR antibodies in order to identify antibodies with high specificity and sensitivity. To address these objectives, we have used multiple immunoassays to determine VDR expression in tissues from several organs from multiple species employing tissues from VDR knockout mice as critical negative controls. Many of the antibodies tested showed nonspecific binding that can account for divergent reports. However, one antibody, identified as D-6, is highly specific and extremely sensitive. The specificity, sensitivity, and versatility of this antibody make it the preferred antibody for identifying VDR expression in target tissues using immunological methods.     

Antibodies to synthetic peptides as probes of acetylcholine receptor structure

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha-chain. Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa). Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9-10 kDa). No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.     

Intracellular third loop-C-terminal tail interaction in prostaglandin EP3beta receptor

The secondary structures of and the interactions between the intracellular domains (the three loops and the C-terminal tail) of the mouse-derived prostaglandin E2 receptor EP3β subtype were investigated using peptides mimicking the domains. The N-termini of the peptides were palmitoylated to anchor on unilamellar vesicles composed of phosphatidylserine, enriched in the cytoplasmic leaflet of mammalian plasma membranes. Circular dichroism spectroscopy revealed that the peptides corresponding to the intracellular third loop (i3) and the C-terminus (C-term) assumed β-sheet and associated α-helical structures, respectively. A structural change was observed when i3 was mixed with C-term, indicating an interaction between them. Fluorescence experiments showed that i3 suppressed the self-association of C-term, confirming the interaction. These results demonstrate for the first time specific interaction between the intracellular third loop and the C-terminus. A model is proposed for the activation of the receptor.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Overexpressed chloride intracellular channel protein CLIC4 (p64H1) is an essential component of novel plasma membrane anion channels

Chloride intracellular channel protein CLIC4 is a putative organellar anion channel or channel regulator with an unusual dual cytoplasmic and integral membrane localisation. To investigate its contribution to cellular anion channel activity, the protein was overexpressed in stably transfected HEK-293 cells. Patch-clamp recording revealed CLIC4-associated indanyloxyacetic acid-sensitive (IC(50) approximately 100 microM) plasma membrane currents showing mild outward rectification, and novel low conductance (approximately 1pS) CLIC4-associated anion channels were resolved at the single-channel level. The CLIC4-associated channels were inhibited by anti-CLIC4 antibodies, including a monoclonal antibody directed against a FLAG epitope fused to the C-terminus of CLIC4, but only when these were applied to the cytoplasmic (not the external) face of the membrane. CLIC4 is thus an essential molecular component of novel cellular anion channels and the C-terminus of the integral membrane form of CLIC4 is cytoplasmic.     

欧洲型舞毒蛾气味受体基因OrCo的克隆及序列分析

【目的】克隆欧洲型舞毒蛾Lymantria dispar Linnaeus的OrCo气味受体基因,并分析其序列特征,为进一步研究舞毒蛾嗅觉机制提供有益参考。【方法】本研究利用RT-PCR和RACE方法,克隆获得欧洲型舞毒蛾OrCo受体基因cDNA全长序列,将该基因命名为LdisOrCo,在GenBank中的登录号为KF482409。【结果】序列分析结果显示,LdisOrCo开放阅读框全长为1 311 bp,编码436个氨基酸,序列中有7个跨膜区和高度保守的C端区域。序列联配分析表明,LdisOrCo基因的氨基酸序列与近缘种灰翅夜蛾(Spodoptera littoralis Or83b)的同源性高达89%,与鞘翅目台湾黑金龟(Holotrichia plumbea Or83b)同源性高达64%,与已经报道的其他昆虫的嗅觉受体同源性都在60%以上,特别是在C端几乎完全一致。【结论】嗅觉受体OrCo(Olfactory receptor coreceptor)在不同昆虫体内高度保守,克隆欧洲型舞毒蛾OrCo基因可以为进一步研究舞毒蛾气味受体的功能,OrCo基因的进化以及揭秘嗅觉机制奠定基础。     

Allelic divergence at Bα1 pheromone receptor genes of Schizophyllum commune

Abstract The multiallelic mating type locus Bal of Schizophyllum commune encodes a pheromone receptor and putative pheromone genes. A comparison of two alleles encoding receptors that share the same specificity Bα1 was performed using strains of different geographic origin. The amino acid sequence alignment revealed strong conservation of the largest part of the receptor. Only in the distal C-terminus major amino acid was divergence encountered. This C-terminal region of 117 of the total of 639 amino acids was shown to be unnecessary for function in vivo by transformation experiments.     

Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors

The crystal structure of the snake long alpha-neurotoxin, alpha-cobratoxin, bound to the pentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 A overall resolution. The structure unambiguously reveals the positions and orientations of all five three-fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand-binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C-terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin-bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic alpha-neurotoxins for the various receptor species.     

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

Binding of glutamate receptor delta2 to its scaffold protein, Delphilin, is regulated by PKA

The glutamate receptor delta2 (GluRdelta2) is selectively expressed in cerebellar Purkinje cells and plays an important role in motor learning, motor coordination, and long-term depression. Delphilin is identified as a GluRdelta2-interacting protein, selectively expressed in Purkinje cell-parallel fiber synapses, and specifically interacts with the GluRdelta2 C-terminus via its PDZ domain. Here, surface plasmon resonance analyses showed that Delphilin PDZ bound to GluRdelta2 C-terminal peptide (DPDRGTSI), but not to its phosphopeptides (DPDRGphosphoTSI and DPDRGTphosphoSI). We showed the incorporation of phosphate into threonine at -2 (-2T) and serine at -1 (-1S) of GluRdelta2 C-terminus by cAMP-dependent protein kinase (PKA) in vitro. In the experiments using heterologous expression system, Delphilin coimmunoprecipitated with GluRdelta2 was dramatically decreased under the condition with forskolin and isobutylmethylxanthine, which led to cAMP-dependent phosphorylation by PKA. Thus, phosphorylation of -2T and/or -1S of GluRdelta2 C-terminus by PKA may regulate the binding of GluRdelta2 to its scaffolding protein, Delphilin.