TYROSINE PHOSPHORYLATION-DEPENDENT STIMULATION OF AMYLOID PRECURSOR PROTEIN SECRETION BY THE M3 MUSCARINIC ACETYLCHOLINE-RECEPTOR

Stimulation of m1 and m3 muscarinic acetylcholine receptors, which are coupled to phosphoinositide hydrolysis and protein kinase C activatio n, has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs). The effect is mimicked by phorbol esters, which directly activate protein kinase C. Using human embryonic kidne y cells expressing individual muscarinic receptor subtypes, we found t hat stimulation of APPs release by the muscarinic agonist carbachol wa s only partially reduced by a specific inhibitor of protein kinase C ( the bisindolylmaleimide GF 109203X), while the response to phorbol 12- myristate 13-acetate (PMA) was abolished. The increase in APPs release elicited by carbachol and PMA was accompanied by elevated tyrosine ph osphorylation of several proteins and reduced by tyrosine kinase inhib itors; GF 109203X significantly reduced the stimulation of tyrosine ph osphorylation by carbachol and PMA. Inhibition of protein tyrosine pho sphatases by vanadyl hydroperoxide markedly increased cellular tyrosin e phosphorylation and enhanced APPs release as effectively as PMA and carbachol. Direct phosphorylation of amyloid precursor protein on tyro sine residues following treatment with carbachol, PMA, or vanadyl hydr operoxide was not observed. The results implicate both tyrosine phosph orylation and protein kinase C dependent mechanisms in the regulation of APPs release by G protein-coupled receptors, and suggest that carba chol and PMA increase APPs release from human embryonic kidney cells e xpressing m3 muscarinic receptors via partially divergent pathways tha t converge at a tyrosine phosphorylation-dependent step.