J Biol Chem, Vol. 274, Issue 31, 21701-21706, July 30, 1999

Muscarinic Cholinergic Receptors Activate Both Inhibitory and Stimulatory Growth Mechanisms in NIH3T3 Cells

From the Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622

Activation of G_q protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21^cip1 expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21^cip1 were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.