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The Journal of Neuroscience, April 11, 2007, 27(15):4083-4092; doi:10.1523/JNEUROSCI.5293-06.2007
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Neurobiology of Disease
M1 and M3 Muscarinic Receptors Control Physiological Processing of Cellular Prion by Modulating ADAM17 Phosphorylation and Activity
Moustapha Alfa Cissé,1 Claire Sunyach,1 Barbara E. Slack,2 Abraham Fisher,3 Bruno Vincent,1 andFrédéric Checler1 1Institut de Pharmacologie Moleculaire et Cellulaire, 06560 Valbonne, France, 2Boston University School of Medicine, Boston, Massachusetts 02118, and 3Israel Institute for Biological Research, 74100 Ness-Ziona, Israel
Correspondence should be addressed to Frédéric Checler or Bruno Vincent, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Université Nice Sophia-Antipolis, Unité Mixte de Recherche 607, Equipe Labellisée Fondation pour la Recherche Médicale, 660 Route des Lucioles, 06560 Valbonne, France. Email: checler{at}ipmc.cnrs.fr or Email: vincentb{at}ipmc.cnrs.fr
The cellular prion protein (PrPc) undergoes a physiological processing yielding the N-terminal fragment referred to as N1, the production of which can be constitutive or protein kinase C regulated. We show that activation of endogenous muscarinic receptors by carbachol and by the M1-selective agonist AF267B increases N1 recovery in an atropine-sensitive manner, in mouse embryonic primary neurons. To identify the muscarinic receptor subtype involved, we used human embryonic kidney HEK293 (HEK) cells stably overexpressing M1, M2, M3, or M4 receptor subtype. Carbachol and the selective M1 agonist AF267B dose dependently increased N1 release by HEK-M3 and HEK-M1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M2 or HEK-M4 cells. We demonstrate that the increase of N1 was not attributable to modified trafficking to the membrane of either PrPc or the disintegrin metalloproteases ADAM10 or ADAM17. Furthermore, we establish that carbachol affects the overall phosphorylation of ADAM17 on its threonine and tyrosine but not serine residues, whereas levels of phosphorylated ADAM9 were not affected. Interestingly, carbachol also increases the hydrolysis of the fluorimetric substrate JMV2770, which mimicked the sequence encompassing the N1 site cleavage and was shown previously to behave as an ADAM protease substrate. Mutations of threonine 735 but not of tyrosine 702 of the ADAM17 cytoplasmic tail abolishes the carbachol-induced increase of N1, ADAM17 phosphorylation, and JMV2770-hydrolyzing activity in M1- and M3-expressing HEK293 cells. Thus, our data provide strong evidence that muscarinic receptor activation increases the physiological processing of PrPc by upregulating the phosphorylation state and activity of ADAM17 protease.
Key words: cellular prion; regulated processing; muscarinic receptors; ADAM17; phosphorylation; HEK293; primary cultured neurons
Received Dec. 7, 2006; revised March 5, 2007; accepted March 6, 2007.
Correspondence should be addressed to Frédéric Checler or Bruno Vincent, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Université Nice Sophia-Antipolis, Unité Mixte de Recherche 607, Equipe Labellisée Fondation pour la Recherche Médicale, 660 Route des Lucioles, 06560 Valbonne, France. Email: checler{at}ipmc.cnrs.fr or Email: vincentb{at}ipmc.cnrs.fr
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