title: M1 and M3 muscarinic receptors control physiological processing of cellular prion by modulating ADAM17 phosphorylation and activity.
abstract: The cellular prion protein (PrP(c)) 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 PrP(c) 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 PrP(c) by upregulating the phosphorylation state and activity of ADAM17 protease.
authors.affiliation: Institut de Pharmacologie Moleculaire et Cellulaire, 06560 Valbonne, France.
authors.forename: Moustapha
authors.initials: M
authors.lastname: Alfa Cissé
authors.forename: Claire
authors.initials: C
authors.lastname: Sunyach
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Slack
authors.forename: Abraham
authors.initials: A
authors.lastname: Fisher
authors.forename: Bruno
authors.initials: B
authors.lastname: Vincent
authors.forename: Frédéric
authors.initials: F
authors.lastname: Checler
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072198:ADAM17 Protein; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011328:Prions / Q000502:physiology*; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D011328:Prions; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D002217:Carbachol; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse