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B Kieffer

Publications and source records attributed to B Kieffer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dynorphin A as a potential endogenous ligand for four members of the opioid receptor gene family.

Dynorphin A is an endogenous opioid peptide that activates the kappa opioid receptor (KOR) with high potency. Some studies also showed that the distribution and functional activity of dynorphin A are not completely correlated with those of KOR, suggesting that dynorphin A may interact with other receptors. To investigate the possibility that dynorphin A may serve as an agonist for other opioid receptors, we took the advantage of the cloning of the three major types of opioid receptors, mu (MOR), delta (DOR) and KOR, and examined their affinity for and their activation by dynorphin A. We used mammalian cells transfected with each of the cDNA clones for the human receptors hMOR, hDOR, hKOR and showed that dynorphin A displaced [3H]-diprenorphine binding with Ki values in the nanomolar range at all three receptors. We also showed that, when hMOR, hDOR or hKOR was coexpressed with a G protein-activated potassium channel in Xenopus oocytes, dynorphin A induced a potassium current with EC50 values in the nanomolar range for all three receptors. Furthermore, we showed that the human hORLI, an opioid receptor-like receptor that has been identified as a novel member of the opioid receptor gene family, displayed dynorphin A binding and functional activation. These results indicate that dynorphin A is capable of binding to and functional activation of all members of the opioid receptor family, suggesting that, as a potential endogenous agonist, its activity in humans may involve interaction with other members of the opioid receptor family in addition to kappa receptors.

Animals↗
PubMed1998

Functional significance of cysteine residues in the delta opioid receptor studied by site-directed mutagenesis.

Previous work suggested that sulfhydryl groups and disulfide bridges have important functions in opioid binding to the delta opioid receptor. The question regarding which cysteines are essential for ligand binding was approached by replacement of cysteine residues in the cloned delta opioid receptor using site-directed mutagenesis. The wild-type and mutant receptors were expressed stably in Chinese hamster ovary cells. The two extracellular cysteine residues and the six located in transmembrane domains were mutated to serine or alanine, one at a time. Replacement of either of the extracellular cysteines produced a receptor devoid of delta agonist and antagonist binding activity. Immunofluorescence cytochemistry, performed with anti delta opioid receptor antibodies in washed cell monolayers in one of these mutants (Cys-Ser121), and immunoblots, performed on cell extracts, indicate that the receptor was expressed and seems to be associated with the cell membrane. The existence of an essential extracellular disulfide bridge, previously postulated by analogy to other G protein coupled receptors, is strongly supported by our results. Replacement of any one of the six transmembrane cysteines was virtually without effect on the ability of the receptor to bind delta agonists and antagonists. Since there is strong evidence that the transmembrane domains are involved in ligand binding, these results suggest that the cysteine residues, even those near or at the binding site, are not essential for receptor binding. Furthermore, these results support the idea that the striking effects of sulfhydryl reagents on ligand binding of opioid receptors are likely to be due to steric hindrance by the large moieties transferred to the sulfhydryl groups of cysteine residues by these reagents.

Alanine↗
PubMed1997

Quantitative autoradiographic mapping of mu-, delta- and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene.

Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any mu-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. Mu-, delta-, kappa1- and total kappa-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin), [3H]DELTI (D-Ala2 deltorphinI), [3H]CI-977 and [3H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, mu-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from mu-knockout mice there were no detectable mu-receptors in any brain regions and no evidence for mu-receptors derived from another gene. Delta-, kappa1- and total kappa-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in kappa- or delta-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in delta- and kappa-receptors throughout the brains of animals deficient in mu-receptors.

Animals↗
PubMed1997

Characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells.

The cDNAs encoding human delta (hDOR), kappa (hKOR) and micro (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.

Analgesics, Opioid↗
PubMed1996

The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site.

Opioids are cationic compounds that mediate their biological action through three highly homologous receptors (mu, delta, and kappa) known to belong to the G protein-coupled receptor (GPR) family. The third putative transmembrane domain of opioid receptors contains a conserved aspartate residue that is typically found in biogenic amine binding GPRs and is generally believed to form an ion pair with the cationic neurotransmitters. Using site-directed mutagenesis, we investigated the possibility of an identical role for this residue (Asp128) in the mouse delta-opioid receptor. Removal of the carboxylate group via an aspartate-to-alanine mutation did not modify binding affinity of a representative set of opioid compounds, including bremazocine, diprenorphine, naloxone, Tyr-D-Thr-Gly-Phe-Leu-Thr, [D-Ala2,D-Leu5]enkephalin, cyclic[D-penicillamine2,D-penicillamine5]enkephalin, deltorphin II, (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di-methyl-1- piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide, and naltrindole. It nevertheless decreased receptor expression level and affected the binding of three agonists ([D-Ala2,D-Leu5]enkephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr, and (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di- methyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide) when the receptor was under Na(+)-induced low affinity state. On the other hand, the aspartate-to-asparagine mutation strongly impaired the binding of all of the above ligands and highlighted differential modes of interaction for alkaloids and peptides. Finally, removal of the homologous carboxylate group in the mouse mu receptor had distinct effects because it dramatically reduced the binding potency of some, but not all, tested ligands. Taken together, these results demonstrate that (i) the direct ligand/receptor interaction previously demonstrated for the beta-adrenergic receptor does not take place in the delta receptor, (ii) Asp128 nevertheless contributes to stabilization of the spatial conformation of the binding pocket, and (iii) these conclusions cannot be extended to the closely related mu receptor.

Amino Acid Sequence↗
PubMed1996

Functional selectivity of orphanin FQ for its receptor coexpressed with potassium channel subunits in Xenopus laevis oocytes.

An opioid-like receptor has been cloned by several groups of researchers and recently shown to be activated by an endogenous heptadecapeptide termed orphanin FQ (or nociceptin). We isolated the corresponding mouse cDNA and coexpressed it in Xenopus laevis oocytes with the potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (CIR, rcKATP). Orphanin FQ evoked potassium currents, with 50% of the maximal effect at approximately 1 nM; [Tyr1]orphanin FQ was equally effective, and des-pheorphanin FQ was without activity. Dynorphin A, dynorphin(1-9), dynorphin(1-13), and alpha-neoendorphin were > 100 times less potent, and other agonists active at mu-, delta-, and kappa-opioid receptors had no effect. Naloxone (1 microM) and norbinaltorphimine (1 microM) had no antagonist action. Conversely, oocytes expressing kappa receptors responded to dynorphin (half-maximal concentration, 0.3 nM) but not to orphanin FQ. Thus, both kappa and orphanin FQ receptors readily couple to potassium channels, but the highly selective activation by dynorphin and orphanin FQ is consistent with distinct functional pathways in vivo.

Amino Acid Sequence↗
PubMed1995

Identification of kappa- and delta-opioid receptor transcripts in immune cells.

To investigate the role of opioids as direct modulators of the immune response, we have searched for expression of the recently cloned delta, mu and kappa opioid receptors in immune cells. We have devised a reverse transcriptase-polymerase chain reaction strategy which specifically detects a region spanning putative transmembrane regions 2 to 7 for each transcript in both human and mouse immune cells. In human peripheral blood lymphocyte and monocyte preparations, delta was undetectable while the kappa transcript was present. The analysis of human cell lines revealed low but significant levels of delta opioid receptor transcripts in T, B or monocyte cell lines while the kappa transcript was found in B cell lines only. Investigation of murine cells showed the presence of transcript for the delta receptor in splenocytes and in some T and B cell lines. Unexpectedly, no expression of the mu receptor was detected. Sequence analysis of PCR products demonstrated nucleotide identity between immune and neuronal transcripts, indicating that they derive from the same genes. In conclusion, our results lead to the identification of kappa and delta opioid receptor transcripts in immune cells.

Animals↗
PubMed1995

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

Adenylyl Cyclases↗
PubMed1994

Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins.

The cell surface antigen CD59 is an inhibitor of complement-mediated lysis and a member of the Ly6 superfamily (Ly6SF) of cysteine-rich cell-surface molecules whose sequences are related to those of snake venom neurotoxins. The three-dimensional solution structure of a recombinant form of the extracellular region of the molecule (residues 1-70 of the mature protein; sCD59) has been solved by 2D NMR methods. sCD59 is a relatively flat, disk-shaped molecule consisting of a two-standed beta-sheet finger loosely packed against a protein core formed by a three-stranded beta-sheet and a short helix. Structure calculations allowed an unambiguous assignment of the disulfide-bonded cysteine pairs as 3-26, 6-13, 19-39, 45-63, and 64-69. The topology of sCD59 is similar to that of the snake venom neurotoxins and consistent with an evolutionary relationship existing between the Ly6SF and the neurotoxins.

Amino Acid Sequence↗
PubMed1994

Delta-opioid receptor gene expression in the mouse forebrain: localization in cholinergic neurons of the striatum.

Opioid peptides and opioid receptors, particularly the delta receptor, are abundant in the striatum where they contribute to the neuronal interactions, and are involved in various behavioral effects. The recent cloning of the delta-opioid receptor now allows the identification of the striatal neurons that express it, and that are direct targets of endogenous opioid peptides such as enkephalins. In this context, we have used in situ hybridization histochemistry to determine the distribution of the delta-opioid receptor messenger RNA in the forebrain, and especially the phenotype of the neurons expressing the delta-opioid receptor gene in the striatum. We show that the topgraphy of the neurons containing the delta-opioid receptor messenger RNA is similar to the topography of the neurons containing the choline acetyltransferase messenger RNA in the mouse forebrain. Comparison of adjacent serial sections demonstrates that the delta-opioid receptor gene is indeed expressed exclusively in cholinergic interneurons in the striatum. As these neurons also selectively express the substance P receptor gene, our data suggest that the striatal cholinergic interneurons are a common link in the interactions between the two striatal efferent populations, namely enkephalin and substance P neurons.

Animals↗
PubMed1994

Structural studies of two antiaggregant RGDW peptides by 1H and 13C NMR.

The structural features of Arg-Gly-Asp-related sequences have been investigated by 1H and 13C NMR. Two linear peptides which inhibit platelet aggregation with a high efficiency have been studied: D-Arg-Gly-Asp-Trp and L-Arg-Gly-Asp-Trp. Analysis of pH titration effects, amide proton exchange rates and inter-proton distances obtained from ROESY spectra suggest that these small fragments predominantly adopt a type II' beta-turn structure in solution. Folding features of a non-active cyclic peptide based on the same sequence (cyclo-[Arg-Gly-Asp-Trp]2) have also been investigated. The biological relevance of these structures is discussed.

Amino Acid Sequence↗
PubMed1994

The human delta-opioid receptor: genomic organization, cDNA cloning, functional expression, and distribution in human brain.

We have used the mouse delta-opioid receptor (mDOR) cDNA to isolate the mDOR gene and its human homologue. In both species the coding region is interrupted by two introns with conserved exon-intron boundaries located after transmembrane domains 1 and 4. Using the polymerase chain reaction and primers based on the sequence of the cloned human delta-opioid receptor (hDOR) gene, we have obtained a full length cDNA encoding the hDOR from SH-SY5Y neuroblastoma cells. The cDNA sequence is 100% identical to the cloned human genomic sequence and 94% identical to the mouse sequence at the protein level. When expressed in COS cells, hDOR displays nanomolar affinities for delta-selective ligands, whereas the affinities for mu- and kappa-selective ligands are in the micromolar range. The delta agonists [D-Ala2, D-Leu5]enkephalin, cyclic [D-penicillamine2,D-penicillamine5]enkephalin, and BW373U86 efficiently decrease forskolin-induced cAMP levels in hDOR-expressing COS cells, indicating functional coupling of the receptor. The distribution of hDOR mRNA in human brain was investigated using delta-selective reverse transcription-polymerase chain reaction amplification, followed by Southern hybridization with a delta-specific probe. The transcript is found in cortical areas, including olfactory bulb, hippocampus, and amygdala, as well as in basal ganglia and hypothalamus. No expression is detected in internal globus pallidus, thalamus, any investigated brainstem structure, or pituitary gland. Taken together, our results indicate similar structural, pharmacological, functional, and anatomical properties for the hDOR and the mDOR and therefore support the use of rodent models for the study of these receptors in opioid function.

Aged↗
PubMed1993

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Amino Acid Sequence↗
PubMed1993

Structural and dynamic studies of two antigenic loops from haemagglutinin: a relaxation matrix approach.

We have investigated the dynamics and structural behaviour of two antigenic peptides using 1H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a beta-turn in both structures.

Amino Acid Sequence↗
PubMed1990

Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae.

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.

Amino Acid Sequence↗