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Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction.
J Biol Chem. 1993 Aug 25; 268(24):18280-5.JB

Abstract

We previously reported that transfection of antisense OBCAM (opioid-binding cell adhesion molecule) cDNA into NG108-15 neuroblastoma x glioma hybrid cells, which contain delta-opioid receptors, results in greatly reduced opioid binding (Ann, D. K., Hasegawa, J., Ko, J. L., Chen, S. T., Lee, N. M., and Loh, H. H. (1992) J. Biol. Chem. 267, 7921-7926. Here we report that these cells show altered coupling between opioid receptors and G-proteins. G-proteins were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for Gi2 and Go alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into a 39-41-kDa protein with the same electrophoretic mobility as Gi2 and Go alpha subunits. This band, which was also a pertussis toxin (PTX) substrate, exhibited decreased CTX-induced ADP-ribosylation in membranes of cells treated chronically with D-Ala2-D-Leu5-enkephalin (DADLE). In cells transfected with antisense cDNA for OBCAM, labeling of this band was also decreased, compared with either sense-transfected or untransfected cells. DADLE inhibition of adenylyl cyclase and DADLE stimulation of GTPase were also greatly impaired in antisense cells, as well as GTP and GppNHp inhibition of basal and forskolin-stimulated adenylyl cyclase. These results provide further evidence for a role of OBCAM in opioid receptor function.

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Authors+Show Affiliations

Govitrapong P
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Zhang X
No affiliation info available
Loh HH
No affiliation info available
Lee NM
No affiliation info available

MeSH

Adenosine Diphosphate RiboseAdenylate Cyclase ToxinAdenylyl CyclasesAnimalsCarrier ProteinsCell Adhesion MoleculesCell MembraneCholera ToxinDNA, AntisenseElectrophoresis, Polyacrylamide GelEnkephalin, Leucine-2-AlanineGPI-Linked ProteinsGTP PhosphohydrolasesGTP-Binding ProteinsGliomaHybrid CellsKineticsMembrane ProteinsMiceMolecular WeightNADNaloxoneNeuroblastomaPertussis ToxinRatsReceptors, Opioid, deltaTransfectionTumor Cells, CulturedVirulence Factors, Bordetella

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8394363

Citation

Govitrapong, P, et al. "Transfection of NG108-15 Cells With Antisense Opioid-binding Cell Adhesion Molecule cDNA Alters Opioid receptor-G-protein Interaction." The Journal of Biological Chemistry, vol. 268, no. 24, 1993, pp. 18280-5.
Govitrapong P, Zhang X, Loh HH, et al. Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction. J Biol Chem. 1993;268(24):18280-5.
Govitrapong, P., Zhang, X., Loh, H. H., & Lee, N. M. (1993). Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction. The Journal of Biological Chemistry, 268(24), 18280-5.
Govitrapong P, et al. Transfection of NG108-15 Cells With Antisense Opioid-binding Cell Adhesion Molecule cDNA Alters Opioid receptor-G-protein Interaction. J Biol Chem. 1993 Aug 25;268(24):18280-5. PubMed PMID: 8394363.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction. AU - Govitrapong,P, AU - Zhang,X, AU - Loh,H H, AU - Lee,N M, PY - 1993/8/25/pubmed PY - 1993/8/25/medline PY - 1993/8/25/entrez SP - 18280 EP - 5 JF - The Journal of biological chemistry JO - J Biol Chem VL - 268 IS - 24 N2 - We previously reported that transfection of antisense OBCAM (opioid-binding cell adhesion molecule) cDNA into NG108-15 neuroblastoma x glioma hybrid cells, which contain delta-opioid receptors, results in greatly reduced opioid binding (Ann, D. K., Hasegawa, J., Ko, J. L., Chen, S. T., Lee, N. M., and Loh, H. H. (1992) J. Biol. Chem. 267, 7921-7926. Here we report that these cells show altered coupling between opioid receptors and G-proteins. G-proteins were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for Gi2 and Go alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into a 39-41-kDa protein with the same electrophoretic mobility as Gi2 and Go alpha subunits. This band, which was also a pertussis toxin (PTX) substrate, exhibited decreased CTX-induced ADP-ribosylation in membranes of cells treated chronically with D-Ala2-D-Leu5-enkephalin (DADLE). In cells transfected with antisense cDNA for OBCAM, labeling of this band was also decreased, compared with either sense-transfected or untransfected cells. DADLE inhibition of adenylyl cyclase and DADLE stimulation of GTPase were also greatly impaired in antisense cells, as well as GTP and GppNHp inhibition of basal and forskolin-stimulated adenylyl cyclase. These results provide further evidence for a role of OBCAM in opioid receptor function. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/8394363/Transfection_of_NG108_15_cells_with_antisense_opioid_binding_cell_adhesion_molecule_cDNA_alters_opioid_receptor_G_protein_interaction_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(17)46841-4 DB - PRIME DP - Unbound Medicine ER -
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