Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Ectodomain shedding of the Notch ligand Jagged1 is mediated by ADAM17, but is not a lipid-raft-associated event
AU - Parr-Sturgess, Catherine A
AU - Rushton, David J
AU - Parkin, Edward T
PY - 2010
Y1 - 2010
N2 - Notch signalling is an evolutionarily conserved pathway involved in cell-fate specification. The initiating event in this pathway is the binding of a Notch receptor to a DSL (Delta/Serrate/Lag-2) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalysed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch-binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signalling. In the present study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a GPI (glycosylphosphatidylinositol) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid-raft-independent manner, and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding.
AB - Notch signalling is an evolutionarily conserved pathway involved in cell-fate specification. The initiating event in this pathway is the binding of a Notch receptor to a DSL (Delta/Serrate/Lag-2) ligand on neighbouring cells triggering the proteolytic cleavage of Notch within its extracellular juxtamembrane region; a process known as proteolytic 'shedding' and catalysed by members of the ADAM (a disintegrin and metalloproteinase) family of enzymes. Jagged1 is a Notch-binding DSL ligand which is also shed by an ADAM-like activity raising the possibility of bi-directional cell-cell Notch signalling. In the present study we have unequivocally identified the sheddase responsible for shedding Jagged1 as ADAM17, the activity of which has previously been shown to be localized within specialized microdomains of the cell membrane known as 'lipid rafts'. However, we have shown that replacing the transmembrane and cytosolic regions of Jagged1 with a GPI (glycosylphosphatidylinositol) anchor, thereby targeting the protein to lipid rafts, did not enhance its shedding. Furthermore, the Jagged1 holoprotein, its ADAM-cleaved C-terminal fragment and ADAM17 were not enriched in raft preparations devoid of contaminating non-raft proteins. We have also demonstrated that wild-type Jagged1 and a truncated polypeptide-anchored variant lacking the cytosolic domain were subject to similar constitutive and phorbol ester-regulated shedding. Collectively these data demonstrate that Jagged1 is shed by ADAM17 in a lipid-raft-independent manner, and that the cytosolic domain of the former protein is not a pre-requisite for either constitutive or regulated shedding.
KW - ADAM Proteins
KW - Alkaline Phosphatase
KW - Base Sequence
KW - Calcium-Binding Proteins
KW - Cell Membrane
KW - Conserved Sequence
KW - Cytosol
KW - Evolution, Molecular
KW - HEK293 Cells
KW - Humans
KW - Intercellular Signaling Peptides and Proteins
KW - Membrane Microdomains
KW - Membrane Proteins
KW - Peptide Fragments
KW - RNA, Small Interfering
KW - Transfection
UR - http://www.scopus.com/inward/record.url?scp=78649583249&partnerID=8YFLogxK
U2 - 10.1042/BJ20100321
DO - 10.1042/BJ20100321
M3 - Journal article
C2 - 20819075
VL - 432
SP - 283
EP - 294
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 2
ER -