Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.

REFERENCES

Artavanis-Tsakonas, Rand
M. D.
,
Lake
R. J.
(
1999
).
Notch signaling: Cell fate control and signal integration in development.
Science
284
,
770
776
Bader
D.
,
Masake
T.
,
Fischman
D. A.
(
1982
).
Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro.
J. Cell Biol
95
,
763
770
Brou
C.
,
Logeat
F.
,
Gupta
N.
,
Bessia
C.
,
LeBail
O.
,
Doedens
J. R.
,
Cumano
A.
,
Roux
P.
,
Black
R. A.
,
Israel
A.
(
2000
).
A novel proteolytic cleavage involved in Notch signaling: The role of the dieintegrin-metalloprotease TACE.
Mol. Cell
5
,
207
216
Dorsky
R. I.
,
Chang
W. S.
,
Rapaport
D. H.
,
Harris
W.
(
1997
).
Regulation of neuronal diversity in the Xenopus retina by Delta signaling.
Nature
385
,
67
–.
Fitzgerald
K.
,
Greenwald
I.
(
1995
).
Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo.
Development
121
,
4275
4282
Gray
G. E.
,
Mann
R. S.
,
Mitsiadis
E.
,
Henrique
D.
,
Carcangiu
M.
,
Banks
A.
,
Leiman
J.
,
Ward
D.
,
Ish-Horowitz
D.
,
Artavanis-Tsakonas
S.
(
1999
).
Human ligands of the Notch receptor.
Am. J. Pathol
154
,
785
794
Greenwald
I.
,
Rubin
G. M.
(
1992
).
Making a difference: the role of cell-cell interaction in establishing separate identities for equivalent cells.
Cell
68
,
271
–.
Han
W.
,
Ye
Q.
,
Moore
M.
(
2000
).
A soluble form of human Delta-like-1 inhibits differentiation of hematopoietic progenitor cells.
Blood
95
,
1616
1625
Heitzler
P.
,
Simpson
P.
(
1991
).
The choice of cell fate in the epidermis of Drosophila.
Cell
64
,
1083
1092
Hukriede
N. A.
,
Gu
Y.
,
Fleming
R. J.
(
1997
).
A dominant-negative form of Serrate acts as a general antagonist of Notch activation.
Development
124
,
3427
3437
Jarriault
S.
,
Brou
C.
,
Logeat
F.
,
Schroeter
E.
,
Kopan
R.
,
Israel
A.
(
1995
).
Signaling downstream of activated mammalian Notch.
Nature
377
,
355
358
Kopan
R.
,
Nye
J. S.
,
Weintraub
H.
(
1994
).
The intracellular domain of mouse Notch: aconstitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD.
Development
120
,
2385
2396
Li
L.
,
Milner
L. A.
,
Deng
Y.
,
Iwata
M.
,
Banta
A.
,
Graf
L.
,
Marcovina
S.
,
Friedman
C.
,
Trask
B. J.
,
Hood
L.
, et al. 
. (
1998
).
The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1.
Immunity
8
,
43
55
Lindsell
C. E.
,
Shawber
C. J.
,
Boulter
J.
,
Weinmaster
G.
(
1995
).
Jagged: A mammalian ligand that activates Notch1.
Cell
80
,
909
917
Luo
B.
,
Aster
J. C.
,
Hasserjian
R. P.
,
Kuo
F.
,
Sklar
J.
(
1997
).
Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.
Mol. Cell. Biol
17
,
6057
6067
Morrison
S. J.
,
Perez
S. E.
,
Qiao
Z.
,
Verdi
J. M.
,
Hicks
C.
,
Weinmaster
G.
,
Anderson
D. J.
(
2000
).
Transient Notch activation initiates an irreversible switch form neurogenesis to gliogenesis by neural crest stem cells.
Cell
101
,
499
510
Mumm
J. S.
,
Schroeter
E. H.
,
Saxena
M. T.
,
Griesemer
A.
,
Tian
X.
,
Pan
D. J.
,
Ray
W. J.
,
Kopan
R.
(
2000
).
A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1.
Mol. Cell
5
,
197
206
Parks
A. L.
,
Klueg
K. M.
,
Stout
J. R.
,
Muskavitch
M. A. T.
(
2000
).
Ligand endocytosis drives receptor dissociation and activation in the Notch pathway.
Development
127
,
1373
1385
Qi
H.
,
Rand
M. D.
,
Wu
X.
,
Sestan
N.
,
Wang
W.
,
Rakic
P.
,
Xu
T.
,
Artavanis-Tsakonas
S.
(
1999
).
Processing of the Notch ligand Delta by the metalloprotease Kuzbanian.
Science
283
,
91
94
Rand
M. D.
,
Grimm
L. M.
,
Artavanis-Tsakonas
S.
,
Patriub
V.
,
Blacklow
S. C.
,
Sklar
J.
,
Aster
J. C.
(
2000
).
Calcium depletion dissociates and activates heterodimeric Notch receptors.
Mol. Cell Biol
20
,
1825
1835
Schroeter
E. H.
,
Kisslinger
J. A.
,
Kopan
R.
(
1998
).
Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.
Nature
393
,
382
386
Struhl
G.
,
Adachi
A.
(
1998
).
Nuclear access and action of Notch in vivo.
Cell
93
,
649
660
Sun
X.
,
Artavanis-Tsakonas
S.
(
1997
).
Secreted forms of Delta and Serrate define antagonists of Notch signaling in Drosophila.
Development
124
,
3439
3448
Tax
F. E.
,
Yeargers
J. J.
,
Thomas
J. H.
(
1994
).
Sequence of C. elegans Lag-2 reveals a cell signaling domain shared with Delta and Serrate of Drosophila.
Nature
368
,
150
154
Varnum-Finney
B.
,
Purton
L. E.
,
Yu
M.
,
Brashem-Stein
C.
,
Flowers
D.
,
Staats
S.
,
Moore
K. A.
,
Le Roux
I.
,
Mann
R.
,
Gray
G.
, et al. 
. (
1998
).
The Notch ligand, Jagged-1, influences the development of primitive hematopoietic cells.
Blood
91
,
4084
4091
Wang
S.
,
Sdrulla
A. D.
,
diSibio
G.
,
Bush
G.
,
Nofziger
D.
,
Hicks
C.
,
Weinmaster
G.
,
Barres
B. A.
(
1998
).
Notch Receptor Activation Inhibits Oligodendrocyte Differentiation.
Neuron
21
,
63
75
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