In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand Bride of sevenless (Boss), which is expressed on the neighboring R8 cell. Boss is an unusual ligand of a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail. Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist. Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, we used oligomerized forms of the Boss extracellular domain to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo. Oligomerization was achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of Lac repressor. Binding of these multivalent proteins to Sevenless could be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization. However, neither R8-specific or ubiquitous expression of multivalent Exboss ligands rescued the boss phenotype. Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction. Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization.

REFERENCES

Alberti
S.
,
Oehler
S.
,
von Wilcken-Bergmann
B.
,
Muller-Hill
B.
(
1993
).
Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.
EMBO J
12
,
3227
3236
Banerjee
U.
,
Renfranz
P. J.
,
Hinton
D. R.
,
Rabin
B. A.
,
Benzer
S.
(
1987
).
The Sevenless+protein is expressed apically in cell membranes of developing Drosophila retina; it is not restricted to cell R7.
Cell
51
,
151
158
Basler
K.
,
Hafen
E.
(
1988
).
Control of photoreceptor cell fate by the sevenless protein requires a functional tyrosine kinase domain.
Cell
54
,
299
311
Basler
K.
,
Hafen
E.
(
1989
).
Dynamics of Drosophila eye development and temporal requirements of sevenless expression.
Development
107
,
723
731
Basler
K.
,
Hafen
E.
(
1989
).
Ubiquitous expression of sevenless: position-dependent specification of cell fate.
Science
243
,
931
934
Basler
K.
,
Christen
B.
,
Hafen
E.
(
1991
).
Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye.
Cell
64
,
1069
1081
Blondel
A.
,
Bedouelle
H.
(
1991
).
Engineering the quaternary structure of an exported protein with a leucine zipper.
Protein Eng
4
,
457
461
Bowtell
D. D.
,
Simon
M. A.
,
Rubin
G. M.
(
1988
).
Nucleotide sequence and structure of the sevenless gene of Drosophila melanogaster.
Genes Dev
2
,
620
634
Bowtell
D. D.
,
Simon
M. A.
,
Rubin
G. M.
(
1989
).
Ommatidia in the developing Drosophila eye require and can respond to sevenless for only a restricted period.
Cell
56
,
931
936
Bruckner
K.
,
Pasquale
E. B.
,
Klein
R.
(
1997
).
Tyrosine phosphorylation of transmembrane ligands for Eph receptors.
Science
275
,
1640
1643
Brunner
D.
,
Oellers
N.
,
Szabad
J.
,
Biggs
W. H.
,
Zipursky
S. L.
,
Hafen
E.
(
1994
).
A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways.
Cell
76
,
875
88
Cagan
R. L.
,
Krämer
H.
,
Hart
A. C.
,
Zipursky
S. L.
(
1992
).
The bride of sevenless and sevenless interaction: internalization of a transmembrane ligand.
Cell
69
,
393
399
Chou
W. H.
,
Hall
K. J.
,
Wilson
D. B.
,
Wideman
C. L.
,
Townson
S. M.
,
Chadwell
L. V.
,
Britt
S. G.
(
1996
).
Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.
Neuron
17
,
1101
1115
Davis
S.
,
Gale
N. W.
,
Aldrich
T. H.
,
Maisonpierre
P. C.
,
Lhotak
V.
,
Pawson
T.
,
Goldfarb
M.
,
Yancopoulos
G. D.
(
1994
).
Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.
Science
266
,
816
819
Dickson
B.
,
Sprenger
F.
,
Morrison
D.
,
Hafen
E.
(
1992
).
Raf functions downstream of Ras1 in the Sevenless signal transduction pathway.
Nature
360
,
600
603
Dickson
B.
,
Hafen
E.
(
1994
).
Genetics of signal transduction in invertebrates.
Curr. Opin. Gen. Dev
4
,
64
70
Fortini
M. E.
,
Rubin
G. M.
(
1990
).
Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster.
Genes Dev
4
,
444
463
Fortini
M. E.
,
Simon
M. A.
,
Rubin
G. M.
(
1992
).
Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation.
Nature
355
,
559
561
Freeman
M.
(
1996
).
Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye.
Cell
87
,
651
660
Friedman
A. M.
,
Fischmann
T. O.
,
Steitz
T. A.
(
1995
).
Crystal structure of lac repressor core tetramer and its implications for DNA looping.
Science
268
,
1721
1727
Friedman
G. C.
,
O'Leary
D. D.
(
1996
).
Eph receptor tyrosine kinases and their ligands in neural development.
Curr. Opin. Neurobiol
6
,
127
133
Gadella
T. W.
Jr.
,
Jovin
T. M.
(
1995
).
Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.
J. Cell Biol
129
,
1543
1558
Hart
A. C.
,
Krämer
H.
,
van Vactor
D. L. J.
,
Paidhungat
M.
,
Zipursky
S. L.
(
1990
).
Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments.
Genes Dev
4
,
1835
1847
Hart
A. C.
,
Harrison
S. D.
,
van Vactor
D. L. J.
,
Rubin
G. M.
,
Zipursky
S. L.
(
1993
).
The interaction of bride of sevenless with sevenless is conserved between Drosophilavirilis and Drosophila melanogaster.
Proc. Nat. Acad. Sci. USA
90
,
5047
5051
Hart
A. C.
,
Krämer
H.
,
Zipursky
S. L.
(
1993
).
Extracellular domain of the boss transmembrane ligand acts as an antagonist of the sev receptor.
Nature
361
,
732
736
Henkemeyer
M.
,
Orioli
D.
,
Henderson
J. T.
,
Saxton
T. M.
,
Roder
J.
,
Pawson
T.
,
Klein
R.
(
1996
).
Nuk controls pathfinding of commissural axons in the mammalian central nervous system.
Cell
86
,
35
46
Holland
S. J.
,
Gale
N. W.
,
Mbamalu
G.
,
Yancopoulos
G. D.
,
Henkemeyer
M.
,
Pawson
T.
(
1996
).
Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands.
Nature
383
,
722
725
Hughes
P. E.
,
O'Toole
T. E.
,
Ylanne
J.
,
Shattil
S. J.
,
Ginsberg
M. H.
(
1995
).
The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity.
J. Biol. Chem
270
,
12411
12417
Hukriede
N. A.
,
Fleming
R. J.
(
1997
).
Beaded of Goldschmidt, an antimorphic allele of Serrate, encodes a protein lacking transmembrane and intracellular domains.
Genetics
145
,
359
374
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
Ichtchenko
K.
,
Hata
Y.
,
Nguyen
T.
,
Ullrich
B.
,
Missler
M.
,
Moomaw
C.
,
Sudhof
T. C.
(
1995
).
Neuroligin 1: a splice site-specific ligand for beta-neurexins.
Cell
81
,
435
443
Kaplan
D. R.
,
Miller
F. D.
(
1997
).
Signal transduction by the neurotrophin receptors.
Curr. Opin. Cell Biol
9
,
213
221
Krämer
H.
,
Cagan
R. L.
,
Zipursky
S. L.
(
1991
).
Interaction of bride of sevenless membrane-bound ligand and the sevenless tyrosine-kinase receptor.
Nature
352
,
207
212
Krämer
H.
,
Phistry
M.
(
1996
).
Mutations in the Drosophila hook gene inhibit endocytosis of the Boss transmembrane ligand into multivesicular bodies.
J, Cell Biol
133
,
1205
1216
Lemmon
M. A.
,
Bu
Z.
,
Ladbury
J. E.
,
Zhou
M.
,
Pinchasi
D.
,
Lax
I.
,
Engelman
D. M.
,
Schlessinger
J.
(
1997
).
Two EGF molecules contribute additively to stabilization of the EGFR dimer.
EMBO J
16
,
281
294
Lewis
M.
,
Chang
G.
,
Horton
N. C.
,
Kercher
M. A.
,
Pace
H. C.
,
Schumacher
M. A.
,
Brennan
R. G.
,
Lu
P.
(
1996
).
Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.
Science
271
,
1247
1254
Mullins
M. C.
,
Rubin
G. M.
(
1991
).
Isolation of temperature-sensitive mutations of the tyrosine kinase receptor sevenless (sev) in Drosophila and their use in determining its time of action.
Proc. Nat. Acad. Sci. USA
88
,
9387
9391
O'Toole
T. E.
,
Katagiri
Y.
,
Faull
R. J.
,
Peter
K.
,
Tamura
R.
,
Quaranta
V.
,
Loftus
J. C.
,
Shattil
S. J.
,
Ginsberg
M. H.
(
1994
).
Integrin cytoplasmic domains mediate inside-out signal transduction.
J. Cell Biol
124
,
1047
1059
Papatsenko
D.
,
Sheng
G.
,
Desplan
C.
(
1997
).
A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells.
Development
124
,
1665
1673
Perrimon
N.
(
1993
).
The torso receptor protein-tyrosine kinase signaling pathway: an endless story.
Cell
74
,
219
222
Philo
J. S.
,
Wen
J.
,
Wypych
J.
,
Schwartz
M. G.
,
Mendiaz
E. A.
,
Langley
K. E.
(
1996
).
Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit.
J. Biol. Chem
271
,
6895
6902
Rogge
R.
,
Cagan
R.
,
Majumdar
A.
,
Dulaney
T.
,
Banerjee
U.
(
1992
).
Neuronal development in the Drosophila retina: the sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells.
Proc. Nat. Acad. Sci. USA
89
,
5271
5275
Rubin
G. M.
,
Spradling
A. C.
(
1982
).
Genetic transformation of Drosophila with transposable element vectors.
Science
218
,
348
353
Schlessinger
J.
,
Ullrich
A.
(
1992
).
Growth factor signaling by receptor tyrosine kinases.
Neuron
9
,
383
391
Schneuwly
S.
,
Klemenz
R.
,
Gehring
W. J.
(
1987
).
Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia.
Nature
325
,
816
818
Simon
M. A.
,
Bowtell
D. D.
,
Rubin
G. M.
(
1989
).
Structure and activity of the sevenless protein: a protein tyrosine kinase receptor required for photoreceptor development in Drosophila.
Proc. Nat. Acad. Sci. USA
86
,
8333
8337
Spivak-Kroizman
T.
,
Lemmon
M. A.
,
Dikic
I.
,
Ladbury
J. E.
,
Pinchasi
D.
,
Huang
J.
,
Jaye
M.
,
Crumley
G.
,
Schlessinger
J.
,
Lax
I.
(
1994
).
Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation.
Cell
79
,
1015
1024
Sun
X.
,
Artavanis-Tsakonas
S.
(
1996
).
The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands.
Development
122
,
2465
2474
Sun
X.
,
Artavanis-Tsakonas
S.
(
1997
).
Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila.
Development
124
,
3439
3448
Thummel
C. S.
,
Pirrotta
V.
(
1992
).
New pCaSpeR P element vectors.
Dros. Info. Service
71
,
150
–.
Tomlinson
A.
,
Ready
D. F.
(
1986
).
Sevenless: a cell-specific homeotic mutation of the Drosophila eye.
Science
231
,
400
402
Tomlinson
A.
,
Bowtell
D. D.
,
Hafen
E.
,
Rubin
G. M.
(
1987
).
Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila.
Cell
51
,
143
150
van Vactor
D. L. J.
,
Cagan
R. L.
,
Krämer
H.
,
Zipursky
S. L.
(
1991
).
Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.
Cell
67
,
1145
1155
Zipursky
S. L.
,
Rubin
G. M.
(
1994
).
Determination of neuronal cell fate: lessons from the R7 neuron of Drosophila.
Annu. Rev. Neurosci
17
,
373
397
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