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 phosphorylation. In contrast, neither of these responses has been associated with m4AChR signaling. We hypothesized that activation of the m5AChR would alter tyrosine phosphorylation patterns spatially within the cell and in a calcium influx-sensitive manner. CHO cells stably transfected with m4- or m5AChRs were assessed for spatial localization and quantity of phosphotyrosylated proteins in response to receptor activation. Results were confirmed by immunoblot of whole cell lysates and cytosol and membrane fractions. m5AChR activation increased tyrosine phosphorylation in all subcellular compartments; coincubation with CAI, a calcium influx inhibitor, reduced phosphorylation below basal levels. Western blot confirmed the change of phosphotyrosylated proteins of M(r) 70, 85, 120, and 180 kDa in whole and fractionated cells. PLC-gamma, used as a marker of m5AChR activity, was increased in quantity and degree of phosphorylation in CHOm5 cell membranes and microvilli in response to receptor activation. Both the quantitative increase and tyrosine phosphorylation of PLC-gamma in membrane fractions was inhibited by CAI. In contrast, CC treatment of CHOm4 cells reduced tyrosine phosphorylation throughout the cell. CC-stimulation of m5AChR cells caused a calcium influx-sensitive increase in phosphotyrosylated proteins throughout the cell, though predominantly in the membrane and microvilli. Activation of the m5AChR induces tyrosine phosphorylation, whereas activation of the m4AChR inhibited tyrosine phosphorylation below baseline, further demonstrating the dichotomy between signaling of these two AChRs.
Alessandro
R
R
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Pugnaloni
A
A
Biagini
G
G
Kohn
E C
EC
D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis; D008854:Microscopy, Electron; D018721:Muscarinic Agonists / Q000494:pharmacology; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014230:Triazoles / Q000494:pharmacology; D010738:Type C Phospholipases / Q000032:analysis; D014443:Tyrosine / Q000378:metabolism*
D002121:Calcium Channel Blockers; D007527:Isoenzymes; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D014230:Triazoles; D014443:Tyrosine; C062058:carboxyamido-triazole; D002217:Carbachol; D010738:Type C Phospholipases; D051966:Phospholipase C gamma