Cell-free synthesis of opiate binding sites

Cabon, Florence; Rhyner, Thomas; Blanot, Francois; Goujet‐Zalc, C.; Mallet, Jacques; Zalc, B.

doi:10.1016/0197-0186(87)90012-x

Cited by 3 publications

(2 citation statements)

References 11 publications

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“…It appears therefore that p receptors of P 1 striatal membranes are less selective so that a 6 ligand can exhibit a higher potency when competing with [3H]DAG0. This poor selectivity of opioid receptors in the developing brain is somewhat similar to the finding that the in vitro translation product of mRNA extracted from neuroblastoma X glioma NG108-15 cells (possessing only 6 receptors) can bind both 6 and p ligands (Cabon et al, 1987). Moreover, evidence for differences between young (P5) and adult rat brain receptors, including a higher potency of levorphanol to [3H]DPDPE-labeled receptors in the young animals, was reported very recently (Szucs and Coscia, 1990).…”

Section: Discussionsupporting

confidence: 78%

Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?

Barg¹,

Simantov²

1991

Journal of Neurochemistry

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The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 78%

Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?

Barg¹,

Simantov²

1991

Journal of Neurochemistry

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“…In a previous study we described the experimental conditions that provide a convenient assay system for the newly synthesized opioid binding sites after cell-free in vitro translation of the mRNA purified from the NG108-15 cells (Cabon et al, 1987). The NG108-15 cell line is a hybrid derived by fusion of the C6BU 1 rat glioma with the N18TG2 mouse neuroblastoma cell lines (Klee and Nirenberg, 1974;Hamprecht, 1977).…”

Section: In Vitro Translationmentioning

confidence: 99%

Pharmacological and molecular properties of opioid binding sites synthesized in a cell‐free translation system

Cabon

Cupo

Ruiz‐Gayo

et al. 1990

J of Neuroscience Research

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Cell-free translation of mRNA, extracted from NG108-15 cells, was used to examine some properties of the opioid binding sites synthesized in vitro. A monoclonal antiidiotype antibody directed against the delta opioid receptor immunoprecipitated a major band of Mr 51,000. Translational immunoassays of poly[A]+RNA, size fractionated by methylmercury agarose gel electrophoresis, demonstrated that the 51,000 Mr protein specifically immunoprecipitated by the anti-opioid receptor antiidiotype antibodies was coded by a transcript which length was in the 6 to 8 kb range. Displacement binding studies of tritiated ligands (either bremazocine or delta or mu selective peptides) with type selective opioid ligands showed that only one type of opioid binding site was synthesized in vitro. Although the pharmacological profiles of ligands binding to NG108-15 cells were characteristic of the delta receptor type, the de novo synthesized opioid binding site had lost its delta selectivity and showed equal affinity for both the mu and delta but not for the kappa ligands. Similar to our finding using the immunoprecipitation system, size fractionation of the NG108-15 poly[A]+RNA demonstrated that the transcript coding for the "mu-delta" binding site had a length of 6,500 to 7,500 nucleotides.

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TheE. coli EnvY gene encodes a high affinity opioid binding site

Cabon

Morser²,

Parmantier

et al. 1993

Neurochem Res

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In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with the E. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E. coli DNA. A search for opioid binding sites on E. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

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Cell-free synthesis of opiate binding sites

Cited by 3 publications

References 11 publications

Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?

Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?

Pharmacological and molecular properties of opioid binding sites synthesized in a cell‐free translation system

TheE. coli EnvY gene encodes a high affinity opioid binding site

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