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Nuclear Ca2+/calmodulin translocation activated by mu-opioid (OP3) receptor
D Wang1, L M Tolbert, K W Carlson
1Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, 94143-0446, USA.
Abstract:
Previous evidence has suggested a role for calmodulin (CaM) in opioid receptor signaling. We demonstrate here that morphine stimulation of the mu-opioid (OP3) receptor causes rapid CaM translocation to the nucleus in OP3-transfected human embryonic kidney (HEK)-293 cells and in SH-SY5Y human neuroblastoma cells. Ca2+ influx into the cells resulting from OP3 receptor activation was required for nuclear CaM translocation. Moreover, in HEK-OP3 and SH-SY5Y cells, increased nuclear CaM content was associated with enhanced phosphorylation of the nuclear transcription factor cyclic AMP-responsive element-binding protein. This appeared to be mediated by Ca2+/CaM kinases and also by a pathway involving protein kinase C. CaM was previously shown to bind directly to the OP3 receptor and to be released from the plasma membrane on agonist stimulation. To test whether OP3-mediated CaM release contributes to nuclear CaM signaling, we used a mutant OP3 receptor (K273A) with reduced affinity for CaM that fails to release CaM from the plasma membrane. K273A-OP3 activated Ca2+ influx to a similar extent as wild-type OP3; however, CaM translocation to the nucleus was attenuated. These results indicate that OP3-stimulated Ca2+ influx results in nuclear CaM translocation, which appears to be enhanced by simultaneous CaM release by OP3 wild-type receptor from plasma membranes. These results suggest a novel Ca2+/CaM signaling pathway of opioid receptors in the regulation of transcriptional activity.
Insights
Morphine activates mu-opioid receptors, causing calmodulin (CaM) to move into the nucleus. This nuclear CaM enhances gene transcription via Ca2+/CaM kinases and protein kinase C.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Calmodulin (CaM) is implicated in opioid receptor signaling.
- Opioid receptors, particularly the mu-opioid (OP3) receptor, are key targets in pain management and addiction.
- Understanding the molecular mechanisms of opioid receptor signaling is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the role of calmodulin (CaM) in mu-opioid (OP3) receptor signaling.
- To determine the pathway of CaM translocation to the nucleus following OP3 receptor activation.
- To explore the impact of nuclear CaM on gene transcription.
Main Methods:
- Utilized OP3-transfected human embryonic kidney (HEK)-293 cells and SH-SY5Y neuroblastoma cells.
- Stimulated OP3 receptors with morphine and measured CaM translocation using microscopy.
- Assessed Ca2+ influx and protein phosphorylation (CREB) via Western blotting and kinase assays.
- Employed a K273A mutant OP3 receptor to investigate CaM release from the plasma membrane.
Main Results:
- Morphine stimulation of OP3 receptors induced rapid CaM translocation to the nucleus, dependent on Ca2+ influx.
- Increased nuclear CaM correlated with enhanced phosphorylation of cyclic AMP-responsive element-binding protein (CREB).
- CaM release from the plasma membrane by the wild-type OP3 receptor enhanced nuclear CaM translocation, while a mutant receptor impaired this process.
Conclusions:
- Opioid receptor activation triggers a novel Ca2+/CaM signaling pathway.
- Nuclear CaM translocation, influenced by Ca2+ influx and receptor-mediated CaM release, regulates transcriptional activity.
- This pathway offers potential targets for modulating opioid receptor function and gene expression.