REGULATION OF CELLULAR TYROSINE PHOSPHORYLATION BY STIMULATORY AND INHIBITORY MUSCARINIC ACETYLCHOLINE-RECEPTORS

Tyrosine phosphorylation is a key signaling event in transmembrane and cytoplasmic signal transduction. The m5 muscarinic receptor (m5AChR) responds to ligand stimulation with calcium influx and protein phospho rylation. In contrast, neither of these responses has been associated with m4AChR signaling. We hypothesized that activation of the m5AChR w ould alter tyrosine phosphorylation patterns spatially within the cell and in a calcium influx-sensitive manner. CHO cells stably transfecte d with m4- or m5AChRs were assessed for spatial localization and quant ity of phosphotyrosylated proteins in response to receptor activation. Results were confirmed by immunoblot of whole cell lysates and cytoso l and membrane fractions. m5AChR activation increased tyrosine phospho rylation in all subcellular compartments; coincubation with CAI, a cal cium influx inhibitor, reduced phosphorylation below basal levels. Wes tern blot confirmed the change of phosphotyrosylated proteins of M-r 7 0, 85, 120, and 180 kDa in whole and fractionated cells. PLC-gamma, us ed as a marker of m5AChR activity, was increased in quantity and degre e of phosphorylation in CHOm5 cell membranes and microvilli in respons e to receptor activation. Both the quantitative increase and tyrosine phosphorylation of PLC-gamma in membrane fractions was inhibited by CA I. In contrast, CC treatment of CHOm4 cells reduced tyrosine phosphory lation throughout the cell. CC-stimulation of m5AChR cells caused a ca lcium influx-sensitive increase in phosphotyrosylated proteins through out the cell, though predominantly in the membrane and microvilli. Act ivation of the m5AChR induces tyrosine phosphorylation, whereas activa tion of the m4AChR inhibited tyrosine phosphorylation below baseline, further demonstrating the dichotomy between signaling of these two ACh Rs.