Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.
The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.
Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland. adrian_robert.walmsley@pharma.novartis.com
Adrian R
AR
Walmsley
Gregor
G
McCombie
Ulf
U
Neumann
David
D
Marcellin
Rainer
R
Hillenbrand
Anis K
AK
Mir
Stefan
S
Frentzel
D000818:Animals; D016466:CHO Cells; D002540:Cerebral Cortex / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D058851:GPI-Linked Proteins; D006801:Humans; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009185:Myelin Proteins / Q000378:metabolism*; D018992:Myelin-Associated Glycoprotein / Q000378:metabolism; D063308:Myelin-Oligodendrocyte Glycoprotein; D009447:Neuroblastoma / Q000378:metabolism; D000070816:Nogo Receptor 1; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000502:physiology; D020800:Receptor, Nerve Growth Factor; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
D058851:GPI-Linked Proteins; C571326:MOG protein, human; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D063308:Myelin-Oligodendrocyte Glycoprotein; D000070816:Nogo Receptor 1; C560528:OMG protein, human; D011480:Protease Inhibitors; C486580:RTN4R protein, human; D020800:Receptor, Nerve Growth Factor; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D008666:Metalloendopeptidases; D015032:Zinc