M₁and M₃Muscarinic 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 M 1 -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 M 1 , M 2 , M 3 , or M 4 receptor subtype. Carbachol and the selective M 1 agonist AF267B dose dependently increased N1 release by HEK-M 3 and HEK-M 1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M 2 or HEK-M 4 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 M 1 -and M 3 -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.

ADAM17 silencing by adenovirus encoding miRNA-embedded siRNA revealed essential signal transduction by angiotensin II in vascular smooth muscle cells

“…As such, physiologically relevant data such as structure activity relationship can be obtained by expressing exogenous mutant protein at a physiological level, instead of over-expressing mutant in order to compete with wild type function. Requirement of Thr735 phosphorylation for ADAM17 activation by various agonists in distinct cell systems have been demonstrated [34, 35]. ADAM17 T735A but not Y702F showed dominant-negative activity with over-expression [35, 36].…”

“…Requirement of Thr735 phosphorylation for ADAM17 activation by various agonists in distinct cell systems have been demonstrated [34, 35]. ADAM17 T735A but not Y702F showed dominant-negative activity with over-expression [35, 36]. However, Phorbol ester-induced EGF receptor ligand shedding was rescued with the ADAM17 cytoplasmic domain deletion mutant in ADAM17 null fibroblasts [20].…”

ADAM-17: the enzyme that does it all

“…ADAM-10, -12, -15 and -17 were documented as growth factor shedding enzymes (for recent review see (Edwards et al, 2008). GPCR agonists implicated in inter-receptor crosstalk that utilizes ADAM-17 include angiotensin-II (Lautrette et al, 2005), ATP (Yin and Yu, 2009), lysophosphatidic acid (LPA) and thrombin (Gschwind et al, 2003), membrane type bile acid receptor (M-BAR)/TGR5 (Yasuda et al, 2007), endothelin-1 (Sanderson et al, 2006), carbachol (Alfa Cisse et al, 2007), and serotonin (Gooz et al, 2006). It is important to note that the same GPCR agonists can induce cleavage of various substrates depending on the cell type (and probably the cellular context).…”

Natural products as a source of Alzheimer's drug leads

“…Both direct and indirect PKC activation of the non-amyloidogenic pathways has been demonstrated. PKC can directly phosphorylate TACE (TNF-α converting enzyme, ADAM17), 281,282 which is responsible for regulated α-secretase activity, 283,282 or indirectly activate α-secretase through the MAP kinase ERK1/2. 284,285 …”