Development of a hormone-defined cell system for studying GPCR agonist-activated growth control: Delta-opioid receptor and beta$\sb2$-adrenergic receptor cross-regulation alters agonist-activated cell proliferation and JNK/ERK activation patterns

Date of Completion

January 1997


Biology, Molecular|Biology, Neuroscience|Biology, Cell




G-protein coupled receptors (GPCRs), originally considered restricted to neuronal cell functioning, are now known to regulate proliferation in non-neural cells. In this work, a novel mammalian cell culture system was developed for the purpose of systematically investigating the complex molecular signaling underlying morphine-activated growth control. Morphine belongs to a family of drugs that mimic the action of endogenous G-protein coupled receptor (GPCR) agonists known as opioids. New clonal lines of fibroblast cells expressing either $\delta$-opioid receptors (DOR1/3T3DA) or serotonin-5HT$\sb{\rm 2C}$ receptors, cultured continuously in serum-free medium, were developed to eliminate complications associated with commonly used cell lines cultured in animal serum.^ Investigating changes in gene expression in the receptor-transfected cell lines revealed that endogenous $\beta\sb2$-adrenergic receptor mRNA transcription is induced in the DOR1/3T3DA cell line. Because $\delta$-opioid and $\beta\sb2$-adrenergic receptors are known to oppositely regulate adenyl cyclase and existing clinical evidence indicates an interaction between the opioid/adrenergic systems these cells furnish a novel system for studying cross-regulation between opioid and adrenergic signaling pathways. In this new cell line, agonist-activation of each receptor separately modulates proliferation with an opposing effect. The opposing proliferatory responses are accompanied by differential activation of the MAPK isozymes, JNK and ERK. JNK/ERK activities are selectively stimulated in response to chronic, but not acute, agonist treatments. Dual agonist treatment results in opioid/adrenergic signal cross-regulation in which the opioid-generated signal dominates at the expense of the adrenergic-generated signal. The point of convergence between the two signaling pathways is downstream of second messenger and, based on the kinetics of the MAPK response, transcriptional regulation is likely to link drug treatment to modulation of the MAPK cascade.^ Analyses of cell cycle kinetics in response to chronic morphine exposure and withdrawal reveal that morphine inhibits proliferation by slowing passage through G$\sb1$. After morphine withdrawal, following a 6 day treatment, the cells exhibit a large temporary increase in proliferation rate and, like untreated cells, when cell-cell contacts are made cells are shunted into G$\sb0$. A potential mechanism relating how insulin-generated and GPCR-generated molecular signals might converge to alter cell proliferation is discussed in the concluding chapter. ^