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Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid
P Govitrapong1, X Zhang, H H Loh
1Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
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.
Insights
Opioid-binding cell adhesion molecule (OBCAM) plays a crucial role in opioid receptor signaling. Reduced OBCAM levels impair opioid receptor coupling with G-proteins, affecting cellular responses to opioid agonists.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- NG108-15 cells express delta-opioid receptors.
- Previous studies showed reduced opioid binding upon antisense OBCAM cDNA transfection.
- OBCAM's role in opioid receptor signaling requires further elucidation.
Purpose of the Study:
- To investigate the effect of reduced OBCAM on opioid receptor-G protein coupling.
- To determine the impact of OBCAM on signal transduction pathways downstream of opioid receptors.
Main Methods:
- Cholera toxin (CTX)-induced ADP-ribosylation to identify G-proteins.
- Pertussis toxin (PTX) substrate analysis.
- Assessing adenylyl cyclase activity and GTPase activity.
- Utilizing antisense OBCAM cDNA transfected cells.
Main Results:
- Reduced CTX-induced ADP-ribosylation of a 39-41 kDa protein (Gi2/Go alpha subunits) in antisense OBCAM cells and DADLE-treated cells.
- Impaired DADLE inhibition of adenylyl cyclase and stimulation of GTPase in antisense OBCAM cells.
- Impaired GTP and GppNHp inhibition of adenylyl cyclase in antisense OBCAM cells.
Conclusions:
- OBCAM is involved in the coupling of opioid receptors to G-proteins.
- OBCAM influences the functional activity of opioid receptors and downstream signaling pathways.
- These findings highlight OBCAM as a key modulator of opioid receptor signal transduction.