Functional Analysis of the NHR2 Domain Indicates that
Oligomerization of Neuralized Regulates Ubiquitination and
Endocytosis of Delta during Notch Signaling
Sili Liu,a,bJulia Maeve Bonner,a,bSoline Chanet,c,dCosimo Commisso,a,b* Lara C. Skwarek,a,b* François Schweisguth,c,dand
Gabrielle L. Bouliannea,b
The Hospital for Sick Children, Program in Developmental and Stem Cell Biology, Toronto, Ontario, Canadaa; Department of Molecular Genetics, University of Toronto,
Toronto, Ontario, Canadab; Institut Pasteur, Developmental Biology Department, Paris, Francec; and CNRS, URA2578, Paris, Franced
signaling and regulating cell fate through inductive interactions
(7, 23). Notch signaling is induced through direct cell-cell inter-
actions between membrane-bound Notch ligands, Delta, Serrate,
and Lag-2 (DSL), and the Notch receptor on adjacent cells. The
stream target genes (7). Recessive loss-of-function mutations in
ing of an overgrowth of the nervous system at the expense of the
The ubiquitination and endocytosis of receptors and ligands
have been shown to potentiate Notch signaling (32, 33, 54). Cur-
rently, there are two models hypothesizing how ligand endocyto-
chanical force or pulling model suggests that Delta endocytosis
exerts a force on the Delta-Notch complex that alters the confor-
mation and promotes the cleavage of the Notch extracellular do-
main (NECD), which is a critical step in Notch activation (17, 41,
42, 51). The recycling model suggests that the modification of an
a more effective ligand, which will be re-presented to the cell sur-
face (perhaps at a microdomain of the plasma membrane) to ac-
tivate Notch (2, 4, 14, 20, 47, 52). In the signal-sending cell, Neu-
ralized (Neur) (12, 27, 43, 56) and Mindbomb1 (3, 22, 25, 28, 34,
the Notch ligands Delta and Serrate by ubiquitination.
Neur was one of the first five Notch pathway members identi-
role in all three germ layers during embryonic development (10,
18, 46). In addition, Neur is also required for the development of
vertebrates and plays multiple and essential roles in many de-
the adult central and peripheral nervous system, including bristle
sense organ patterning and photoreceptor specification (29, 30,
57). Consistent with its role in embryogenesis and adult neuro-
genesis, Neur is expressed in embryonic neural tissue and in the
region of larval imaginal discs that will give rise to adult sensory
organs (5). Of note, Neuralized is not required for all Notch sig-
naling events, and evidence suggests that Mind bomb, its func-
developmental contexts (28, 34). The presence of either Neur or
Mind bomb in the signal-sending cell appears to be required for
ligand endocytosis (34). In addition to its role in Notch signaling,
Neur was also recently shown to regulate epithelial cell polarity in
the embryo (8).
The neur locus produces two major transcripts, neur-RA and
neur-RC, which give rise to two proteins, NeurPA and NeurPC,
which differ only at their N termini (9). Specifically, NeurPA,
which is the predominant isoform during development (5), con-
tains a phosphoinositide (PIP)-binding motif at the N terminus,
which is required for Delta endocytosis downstream of Delta
ubiquitination by Neur (49). In addition, both isoforms contain
three highly conserved domains, including a carboxyl-terminal
RING domain and two Neuralized homology repeat (NHR) do-
Received 31 May 2012 Returned for modification 20 June 2012
Accepted 28 September 2012
Published ahead of print 8 October 2012
Address correspondence to Gabrielle L. Boulianne, firstname.lastname@example.org.
*Present address: Cosimo Commisso, Department of Biochemistry, New York
University School of Medicine, New York, New York, USA; Lara C. Skwarek,
Department of Molecular and Cell Biology, University of California, Berkeley,
Berkeley, California, USA.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
December 2012 Volume 32 Number 24 Molecular and Cellular Biologyp. 4933–4945 mcb.asm.org
mains (NHR1 and NHR2). The RING domain is both necessary
and sufficient for Neur E3 ubiquitin ligase activity and is required
for the endocytosis of the Notch ligand Delta (27, 43, 56). The
NHR1 domain is a protein-protein interaction module that is re-
quired for Neur to bind Delta: a point mutation in a highly con-
served glycine residue at position 167 disrupts Delta binding (9).
Neur and Serrate (44) is unknown.
have been characterized, the full spectrum of Neur function and
the NHR2 domain. Previous in vitro studies done with mamma-
lian cell cultures demonstrated that the NHR2 domain of Neurl1,
a mouse homologue of Drosophila Neur, mediates the interaction
between Neurl1 and Jagged1 (26), a mouse homologue of Dro-
sophila Serrate. Whether the NHR2 domain is required for Neur
function in vivo and whether this function depends solely on its
interaction with Serrate remain to be determined.
Here we show that Neur binds both Notch ligands via the
NHR1 domain. We also show that the NHR2 domain is required
for Neur-mediated Dl endocytosis and Notch signaling in vivo.
point mutation in a highly conserved glycine residue within the
that Neur can form oligomers, which regulate its ubiquitination
activity and, as a result, ligand internalization.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. pAC-NeurWT was previously
described (49). By using pAC-NeurWT as a template, Neur?NHR1,
Neur?NHR2, and NeurNHR2were amplified by PCR, cloned into the
pENTR vector (Life Technologies, Grand Island, NY), and subsequently
flipped into the pAW vector (Murphy Laboratory Drosophila Gateway
ington, IN) or the pMGF vector (gift from A. McQuibban, Toronto, On-
The NeurG430Eand Neur?NHR1&2mutants were generated by using a
QuikChange Lightning site-directed mutagenesis kit (Agilent Technolo-
gies, Santa Clara, CA). pMT-DlWT-Ndemyc (24) was obtained from the
DGRC (Bloomington, IN). By using pUAST-Ser-myc (gift from C. Deli-
dakis, Heraklion, Crete, Greece) as a template, cDNA encoding Ser was
amplified by PCR, cloned into the pENTR vector (Life Technologies,
Grand Island, NY), and subsequently flipped into the pMGF vector (gift
from A. McQuibban, Toronto, Ontario, Canada) by Gateway cloning
(Life Technologies, Grand Island, NY). pRM-HA-Ubiquitin was a gift
from P. Rørth (48). Oligonucleotide sequences of PCR primers are avail-
able upon request.
Antibodies. Rabbit anti-V5 antibody (Genscript, Piscataway, NJ) was
used to detect V5-tagged Neur proteins at a 1:3,000 dilution for Western
blots and at 1:500 to 1:1,000 dilutions for immunostaining. Mouse anti-
tagged Neur and Ser proteins by Western blotting (1:3,000 dilution) and
immunostaining (1:1,000 dilution). Rabbit anti-myc antibody (Gen-
script, Piscataway, NJ) was used to detect the myc-tagged Dl protein by
Western blotting (1:3,000 dilution). Mouse anti-Dl antibody (C594-9B;
(E7 and JLA20, respectively; DSHB, Iowa City, IA) were used as loading
controls on Western blots (1:1,000 dilution). Guinea pig anti-Sens anti-
body was used to mark sensory organ precursor cells (SOPs) (1:300 dilu-
tion; H. Bellen, Houston, TX). Rabbit anti-Neur antibody was used to
Yeast two-hybrid analysis. A yeast two-hybrid (Y2H) analysis was
performed by using the Clontech Laboratories (Mountain View, CA)
Neur Start-NHR2. The positive-control vectors used were the known in-
teractors Jun and cFOS as well as full-length GAL4 (original constructs
used for cloning were kindly provided by Charles Boone, Toronto, On-
tario, Canada). Negative controls were empty vectors.
Cell culture. Drosophila Kc167 (Kc) cells were cultured at 23°C in
Schneider’s medium (catalog number S9895; Sigma-Aldrich, St. Louis,
MO) supplemented with 10% fetal bovine serum (PAA, Pasching, Aus-
manufacturer’s specifications (Life Technologies, Grand Island, NY). For
pMT-DlWT-NdeMYC, pMTF-Ser, and pRM-HA-Ubiquitin, 500 ?M
CuSO4was used to induce protein expression for 24 h prior to the assay.
Drosophila stocks and transgenics. Wild-type controls were w1118
flies. Other stocks used in this study include the following: neurA101GAL4
(catalog number 6393) and ombGAL4 (catalog number 3605), obtained
breviated UAS-NeurWT); UAS-neurPA5Q-V5(49); and UAS-neurPC-V5.
neurAA8-N67, obtained by the excision of the Neur-RC-specific P element
a deletion of 1,600 bp that removes most of the first exon encoding
NeurPC. Breakpoints were determined by PCR and DNA sequencing
(ACTG; SickKids). UAS-NeurPA?NHR2-V5(abbreviated UAS-Neur?NHR2)
and UAS-NeurPAG430E-V5(abbreviated UAS-NeurG430E) were injected
were collected. Numbers of wild-type (WT) and neurogenic embryos
were scored, and the neurogenic percentage was calculated. Student’s t
bryos that carried two copies of the balancer chromosome, which are
easily identifiable because they are highly disorganized and positive for
green fluorescent protein (GFP). Given this protocol, Mendelian inheri-
tance predicts that one-third of embryos will be homozygous for
neur mutant clones overexpressing versions of V5-tagged Neur were
generated by using hs-flp, tub-Gal4, UAS-GFP; UAS-NeurV5/?; FRT82B
neurIF65/FRT82B tub-Gal80 flies. Mitotic clones were induced by a 45-
min heat shock at 36.5°C during the first-instar larval stage. All crosses
were performed at 25°C.
Immunohistochemistry. For immunostaining, Kc cells were grown
on 25-mm circular coverslips. Cells were fixed at 48 h after transfection
incubated with primary and secondary antibodies, and then mounted in
Dako mounting medium (Dako, Burlington, Ontario, Canada). Images
were acquired on a Quorum Spinning Disk confocal microscope (Quo-
rum, Guelph, Ontario, Canada). For the quantification of localization,
cells were scored for cytoplasmic or membrane localization; a total of
?100 cells were examined per genotype from 3 individual transfections.
To examine localization in embryos, fly crosses were set up in cages on
standard methanol fixation. Immunostaining protocols were the same as
ing third-instar larvae were dissected in cold phosphate-buffered saline
(PBS). Wing imaginal discs were obtained and treated as described above
for cell culture staining.
Endocytosis assay. The endocytosis protocol was modified from a
method described previously by Le Borgne et al. (35). Wing discs were
dissected in Schneider’s Drosophila medium (catalog number S9895;
Sigma-Aldrich, St. Louis, MO) containing 1% fetal bovine serum (PAA,
Pasching, Austria). After dissection, the medium was replaced and sup-
Liu et al.
mcb.asm.orgMolecular and Cellular Biology
plemented with 1 ?g/ml 20-hydroxyecdysone (Sigma-Aldrich, St. Louis,
MO). Wing discs were cultured for 10 min in the presence of the mouse
monoclonal anti-Dl antibody (1:100 dilution) (C594-9B; DSHB, Iowa
City, IA). Following washes, wing discs were fixed and subjected to sec-
ondary antibody treatment.
Western blot analysis and immunoprecipitation. Embryo lysates
were prepared from 0- to 18-h collections. Embryos were dechorionated
and then lysed in 2? SDS loading buffer. For immunoprecipitation (IP)
immunoprecipitation assay (RIPA) buffer with protease inhibitors
(Roche, Basel, Switzerland). The input was precleared with 25 ?l 50%
RIPA buffer plus 50% protein G-Sepharose (Sigma-Aldrich, St. Louis,
MO) for 1 h at 4°C and incubated with 30 ?l 50% RIPA buffer plus 50%
protein G and 3 ?l anti-V5 antibody (Life Technologies, Grand Island,
loading buffer. For the ubiquitination IP, cells were treated with 50 ?M
calpain inhibitor IV (MG-132; American Peptide Company, Sunnyvale,
CA) for 3 h prior to lysis.
that is required for E3-ligase activity and two highly conserved
Neur homology repeats, NHR1 and NHR2 (Fig. 1A). We have
previously shown that the NHR1 domain mediates Neur binding
deletion of the entire NHR1 domain or the mutation of a highly
FIG 1 The NHR2 domain and the conserved Gly430 residue are required for Neuralized function in vivo. (A) Various V5-tagged Neur constructs used in this
showing the expression of V5-tagged Neur driven by neurA101GAL4. (C to F) Lateral views of the embryonic nervous system (shown in green) identified with a
UAS-NeurWToverexpressed) (D) and neurogenic (with UAS-Neur?NHR2[E] and UAS-NeurG430E[F] overexpressed) neurA101GAL4 embryos showing the
was calculated. (H) Results from overexpression experiments in a heterozygous background. The proportion of wild-type embryos from each cross was
NHR2 Domain Regulates Neur Activity
December 2012 Volume 32 Number 24 mcb.asm.org 4935
rupts binding to Dl (9). To date, however, the function of the
NHR2 domain remains unknown. To determine whether the
NHR2 domain is required for Neur function, we generated trans-
genic flies carrying a deletion of the entire NHR2 domain (UAS-
Neur?NHR2) or a mutation at Gly430 (UAS-NeurG430E), which
corresponds to the Gly167 residue found in the NHR1 domain
(9), under the transcriptional control of the GAL4 upstream acti-
vation sequence (UAS) system (Fig. 1A) (6). To ensure that all of
Western blots with embryonic extracts derived from transgenic
flies expressing NeurWT(49), Neur?NHR2, or NeurG430Eusing
neurA101GAL4. We found that all three transgenes were expressed
at comparable levels, with slightly higher levels of expression ob-
served for the NeurG430Eline (Fig. 1B).
embryonic neurogenic phenotype due to the insertion of a P ele-
ment containing the transcriptional activator GAL4 within the
neur promoter. To determine if the NHR2 domain was required
for Neur function, we examined whether the expression of the
of neurA101GAL4 mutant homozygous embryos (Fig. 1C), which
we scored by using an anti-horseradish peroxidase (HRP) anti-
body (50). We observed that 32.91% of embryo progeny of
neurA101GAL4 heterozygotes were neurogenic in the absence of
any transgene (Fig. 1C and G). In the presence of the NeurWT
transgene, only 7.32% of embryos appeared neurogenic (referred
to as rescue) (Fig. 1D and G), as previously shown (49). In con-
trast, both Neur?NHR2and NeurG430Efailed to rescue the neur
embryos appearing neurogenic: 24.46% and 45.10%, respectively
presence of Neur?NHR2(24.46%) was less than that observed for
neurA101GAL4 alone (32.91%), it remained significantly higher
than what we observed with NeurWT(7.32%). This finding sug-
gests that this construct may retain some residual activity albeit
vation that NeurG430resulted in a significant increase in the per-
centage of neurogenic embryos (Fig. 1G) suggests that this con-
Neur in the heterozygous progeny. Consistent with this interpre-
tation, the ectopic expression of NeurG430Ein a heterozygous
background (UAS-NeurG430E/?; neurA101GAL4/?) also resulted
in a similar increase in the percentage of neurogenic embryos,
while the overexpression of NeurWTor Neur?NHR2had no effect
compared to the effect of heterozygous neurA101GAL4 by itself
(Fig. 1H). Altogether, these data indicate that the deletion of the
residue is essential for Neur function during embryonic neuro-
genesis. In addition, our data suggest that the deletion and point
The NHR2 domain is not required for Neur localization at
the plasma membrane or its interaction with Notch ligands.
Given that the NHR2 domain is important for Neur function, we
then asked whether the inability of the NHR2 mutant transgenes
in vivo. We first analyzed the subcellular localization of the trans-
genic protein in embryos using neurA101GAL4 (Fig. 1D?, E?, and
F?) and in wing imaginal discs using ombGAL4 (see Fig. 5J=, K=,
and L=) and found no significant difference between the localiza-
tions of the mutant proteins and WT Neur. NeurWT, Neur?NHR2,
and NeurG430Eseemed to be enriched at the plasma membrane.
In previous studies, we showed that in singly transfected S2
cells, Neur and Delta were found primarily at the plasma mem-
brane (49). We also showed that a mutant form of Neur lacking a
PIP-binding motif was localized primarily to the cytoplasm in
singly transfected cells but could be recruited to the membrane in
the presence of Dl (49). To determine if mutations in the NHR2
be recruited to the membrane in the presence of Delta, we trans-
localized to the plasma membrane, as previously described (49)
(Fig. 2E and O). However, in contrast to what we previously ob-
served for S2 cells, we found that Neur proteins were predomi-
nantly cytoplasmic in the absence of Dl (Fig. 2A to C and O) (9,
49). This discrepancy may reflect differences between Kc and S2
cell lines. Nonetheless, when cells were cotransfected with Dl and
either WT or NHR2 mutant Neur constructs, Neur relocated to
the plasma membrane (Fig. 2F to H and O), demonstrating that
the NHR2 domain is not required for Neur localization to the
plasma membrane when Dl is present. In contrast, and consistent
with our previous findings (9), a mutation of a highly conserved
glycine residue in the NHR1 domain (NeurG167E) disrupts the
interaction between Neur and Dl, leading to the accumulation of
Neur within the cytoplasm (Fig. 2I and O) and a concomitant
retention of Dl at the membrane (Fig. 2I= and O) (9). The intro-
duction of either NeurWTor Neur?NHR2resulted in an increase in
the cytoplasmic Dl level (Fig. 2F= and G=), although Neur?NHR2
was not as efficient as the WT (Fig. 2O). These results are consis-
tent with our rescue data demonstrating that while Neur?NHR2
of NeurG430E(Fig. 2H= and O). These data suggest that the NHR2
domain is not required for Neur to bind to Delta; however, the
that the NHR2 domain is not required for Neur to bind Dl, we
cotransfected Dl and Neur into Kc cells and performed coimmu-
noprecipitation (co-IP) experiments. As was shown previously,
NeurG167Edisrupts Dl binding in this assay. We found that Dl
coimmunoprecipitated with both NHR2 mutants (Fig. 3A, IP
lanes 5 and 6 on the anti-myc blot), further demonstrating that
mutations in NHR2 do not affect the interaction between Neur
previously shown that the NHR1 domain is both necessary and
sufficient to bind to Dl in Drosophila (9). Others have shown that
Drosophila, we first transfected tagged Neur and/or Ser into Kc
was localized to the plasma membrane in singly transfected cells
or the NHR2 mutant, Neur was also concentrated at the plasma
membrane (Fig. 3K to M and O). Similarly to what we observed
dramatic increase in the cytoplasmic Ser level (Fig. 2K=, L=, and
O), while NeurG430Edid not (Fig. 2M= and O). Interestingly,
Liu et al.
mcb.asm.org Molecular and Cellular Biology
NeurG167Eremained cytoplasmic in the presence of Ser (Fig. 2N
brane (Fig. 2N= and O), suggesting that the G167E mutant is not
recruited to the plasma membrane, where it is required to induce
the internalization of Ser. Furthermore, in coimmunoprecipita-
tion experiments with Neur and Ser, we found that both NHR2
mutants of Neur could be coimmunoprecipitated with Ser (Fig.
3B, IP lanes 9 and 10 on the anti-V5 blot) but not with NeurG167E
(Fig. 3B, IP lane 8 on the anti-V5 blot), suggesting that the G167E
mutation in the NHR1 domain disrupts Neur-Ser binding. Of
note, the interaction between WT Neur and Ser in our co-IP ex-
periment appeared to be very weak; however, given that the ex-
and other researchers previously reported an interaction between
NeurWTand Ser using co-IP experiments (16), we speculated that
the weak binding in our experiments might reflect technical dif-
ferences in co-IP protocols. Taken together, our data show that
the NHR2 domain is not required for the plasma membrane lo-
calization of Neur in the presence of either Dl or Ser, nor is it
required to bind to either ligand. In contrast, the conserved
Gly167 residue in the NHR1 domain is required for Neur to bind
to both Dl and Ser in Drosophila.
ubiquitination activity. Given that the NHR2 domain is not re-
quired for Neur to interact with its targets Dl and Ser, we asked
whether the NHR2 domain or the Gly430 residue is required for
Neur E3-ubiquitin ligase activity. To this end, we performed im-
munoprecipitation experiments with cells cotransfected with
Neur-V5, Dl-myc, and hemagglutinin (HA)-ubiquitin. High lev-
precipitate in the presence of NeurWT(Fig. 4A, IP lane 5 on the
anti-HA blot). In contrast, in the presence of NeurC701S, a Neur
mutant that has no E3 ligase activity (56), the levels of ubiquiti-
nated proteins were significantly reduced (Fig. 4A, compare IP
lane 6 to lane 5 on the anti-HA blot). Both the NeurG167Eand
Neur?NHR2mutants appeared to be as efficient at ubiquitination
as NeurWT(Fig. 4A, compare IP lanes 7 and 8 to lane 5 on the
ubiquitinated proteins correspond to Delta, Neur, or other un-
identified partners. Given that NeurG167Edoes not bind to Dl but
transfections (F? to I?). (J to N) Single confocal slices of Kc cells expressing FLAG-Ser (J) or in combination with NeurWT, Neur?NHR2, NeurG430E, or NeurG167E
(K to N for Neur and K= to N= for Ser) and merged images of double transfections (K? to N?). Neur is shown in green. Dl and Ser are shown in red. (O)
Quantification of Kc cells expressing Dl-myc or FLAG-Ser in combination with NeurWT, Neur?NHR2, NeurG430E, or NeurG167E. Percentages of cells showing
either a membrane or cytoplasmic localization were calculated for ligands and Neur variants.
NHR2 Domain Regulates Neur Activity
December 2012 Volume 32 Number 24mcb.asm.org 4937
Neur is autoubiquitinated or has other targets besides Dl. Consis-
tent with this idea, in a similar co-IP experiment, where we ex-
pressed Neur and HA-ubiquitin on their own, we observed high
levels of HA-tagged ubiquitin coimmunoprecipitating with Neur
Dl may not be the only target of Neur. Nonetheless, the ubiquiti-
nation activity was completely abolished in NeurG430E(Fig. 4A,
NHR1 or the NHR2 domain had no effect (Fig. 4A, lane 8 on the
anti-HA blot, and B, lanes 4 and 6 on the anti-HA blot), demon-
strating that the two mutations in NHR2 have distinct effects on
Neur function. Interestingly, when the NHR1 domain was re-
moved from NeurG430E, ubiquitination activity was restored (Fig.
4B, lane 5).
The NHR2 domain is required for Neur-mediated Dl endo-
cytosis in vivo. We have shown that the deletion of the NHR2
was still able to induce Dl and Ser internalization albeit with re-
duced activity. Switching from cultured cells, we wanted to assess
the role of the NHR2 domain in Dl endocytosis in flies, a more
physiologically relevant context. To address this, we first assessed
the effect of the expression of Neur transgenes on Dl localization
throughout the presumptive wing pouch using ombGAL4. Of
the wing vein primordia (Fig. 5E, arrow). The ectopic expression
of NeurWTresulted in an increase in the amount of internalized
in the presence of either Neur?NHR2or NeurG430E(compare Fig.
5G and H to E), suggesting that NHR2 mutant proteins failed to
facilitate Dl endocytosis.
To further confirm that the differences that we saw in the lo-
calization of Dl were due to a defect in endocytosis, we used an
presence of exogenous Neur. Specifically, we used ombGAL4 to
drive the expression of Neur transgenes and examined Dl endo-
cytosis using an anti-Dl antibody generated against the extracel-
lular domain of Dl (DlECD). Consistent with our previously re-
ported observations (49), we found that the ectopic expression of
NeurWTresulted in an increase in Dl-antibody internalization
(Fig. 5J). Neur?NHR2showed significantly decreased levels of Dl-
antibody internalization (Fig. 5K), indicating that the deletion of
minimal levels of internalized Dl were observed for the NeurG430E
mutant (Fig. 5L), since the G430E mutant has no ubiquitination
Neur forms oligomers. RING domain E3 ligases often func-
tion as oligomers (13). Evidence for the oligomerization of Neur
in vivo was obtained by examining the localization of the two
isoforms of Neur (NeurPAand NeurPC) during the asymmetric
asymmetrically at the anterior cortex when SOPs divide along the
anterior-posterior axis to produce a posterior pIIa cell and an
anterior pIIb cell (Fig. 6A) (15, 35). We found that the anterior
NeurPC-specific mutant, neurAA8-N67, that deletes the first exon of
NeurPCspecifically (Fig. 6B), suggesting that the asymmetrical
the minor isoform, is essentially an N-terminal truncation of
NeurPA. Importantly, NeurPClacks a PIP-binding motif that was
previously shown to be required for the plasma membrane asso-
ciation and function of NeurPA(49) and is cytoplasmic in vivo
(data not shown).
We then examined the distributions of V5-tagged NeurPAand
NeurPCexpressed in SOPs using neurA101GAL4 and found that
both NeurPAand NeurPClocalized asymmetrically (Fig. 6C and
neurIF65mutant cells, we observed that NeurPAbut not NeurPC
localized asymmetrically (Fig. 6F and G). We therefore conclude
This observation suggested that NeurPArecruits NeurPCat the
NeurPAdiffers from NeurPCby the presence of a PIP-binding
localization of NeurPA. To do so, we examined the distribution of
NeurPA5Q, a NeurPAversion with a mutated PIP-binding motif
(49). In the presence of endogenous Neur, NeurPA5Qlocalized to
an anterior crescent in the metaphase and segregated into the an-
terior cell (Fig. 6E). However, similarly to NeurPC, this asymmet-
ric distribution of NeurPA5Qwas found to require endogenous
was Neur independent (35). Thus, our analysis of both NeurPC
and NeurPA5Qrevealed that the asymmetric localization of Neur
proteins lacking the N-terminal PIP-binding motif required en-
FIG 3 The G167E mutation in the NHR1 domain disrupts the Ser-Neur in-
teraction. (A) NeurWT, Neur?NHR2, and NeurG430Ecan coimmunoprecipitate
full-length Dl from Kc cell lysates. NeurG167Efailed to coimmunoprecipitate
full-length Dl. (B) Ser can coimmunoprecipitate NeurWT, Neur?NHR2, and
NeurG430Efrom Kc cell lysates but fails to pull down NeurG167E. The input
contains 1/20 of the lysate used for immunoprecipitation.
Liu et al.
mcb.asm.org Molecular and Cellular Biology
in vivo with NeurPCand NeurPA5Qto promote their asymmetric
We next verified the ability of Neur to form oligomers in vitro.
transfected these constructs into Kc cells, and performed anti-V5
immunoprecipitations. We found that FLAG-tagged Neur could
be immunoprecipitated with NeurWT-V5(Fig. 7B, IP lane 3 on the
anti-FLAG blot), further demonstrating that Neur can form
tion, we performed immunoprecipitation experiments with
lysates obtained from Kc cells cotransfected with various wild-
type and truncated Neur constructs (Fig. 7A). We observed a
strong interaction between Neur?NHR1-V5and Neur?NHR1-FLAG
(Fig. 7B, IP lane 7 on the anti-FLAG blot), suggesting that the
NHR1 domain cannot be the sole mediator of oligomerization.
Similarly, we found that the deletion of the NHR2 domain alone
does not affect Neur oligomerization (Fig. 7B, IP lane 9 on the
anti-FLAG blot). We also found that Neur?NHR1-FLAGand
and NHR2 were deleted, the remaining RING domain and the
lane 7 on the anti-FLAG blot). To determine whether the NHR
domains alone are capable of mediating Neur dimerization, we
also performed a direct yeast two-hybrid analysis (Fig. 7D). Re-
sults from two independent trials indicated that Neur can
dimerize via the NHR1 domain (Fig. 7E). Moreover, a con-
struct containing both the NHR1 and NHR2 domains is capa-
ble of binding to the NHR2 domain alone (Fig. 7E). Although
we did not observe an interaction between the NHR1 and
NHR2 domains in Y2H experiments, it is possible that factors
required for the NHR1-NHR2 interaction were missing in the
in vitro system. When we performed immunoprecipitation ex-
periments with lysates obtained from Kc cells cotransfected
with NHR1-V5 (9) and NHR2-FLAG, we found that NHR1 can
FIG4 The G430 residue in the NHR2 domain is required for Neuralized ubiquitination activity. Neur variants and HA-ubiquitin were cotransfected into
Kc cells either with Dl (A) or without Dl (B), followed by anti-V5 immunoprecipitation. The immunoprecipitation of Neur was performed with cells
treated with calpain IV (proteasome inhibitor) for 3 h prior to lysis. (A) In the immunoprecipitation blot, myc-Dl was pulled down with Neur variants
except NeurG167E. HA-tagged ubiquitinated products were present when NeurWT, Neur?NHR2, or NeurG167Ewas expressed. However, the expression of
NeurG430Esignificantly abolished the ubiquitination activity, with a minimum amount of ubiquitinated product being coimmunoprecipitated. (??,
full-length Dl; ???, C-terminal cleavage product of Dl.) (B) In the immunoprecipitation blot, HA-tagged ubiquitinated products were present when
NeurWT, Neur?NHR1, Neur?NHR1G430E, or Neur?NHR2was expressed and were absent when NeurG430Ewas expressed. The input contains 1/20 of the lysate
used for immunoprecipitation. ?, nonspecific bands and staining.
NHR2 Domain Regulates Neur Activity
December 2012 Volume 32 Number 24 mcb.asm.org 4939
interact with NHR2 (Fig. 7F). These data suggest that the NHR
domains are sufficient but not necessary for oligomerization,
and there may be multiple ways for oligomers to be assembled
As shown previously, the Gly430 residue in the NHR2 domain
is essential for Neur ubiquitination activity. To test whether
Gly430 plays a role in protein oligomerization, we cotransfected
NeurG430Ewith NeurWT-FLAGinto Kc cells, followed by anti-V5
immunoprecipitation. We found that the G430E mutant could
still form oligomers with WT Neur but was less efficient than the
(A), UAS-NeurWT(B), UAS-Neur?NHR2(C), and NeurG430E(D) in the developing wing pouch, using ombGAL4 (green), merged with Dl staining (red; shown
separately in panels A= to D=). (E to H) Higher magnification of Dl staining in the wing pouch (white square in panel A=). Dl is expressed at its highest levels in
is largely reduced in the presence of Neur?NHR2. (L) Dl is not internalized at a detectable level when NeurG430Ewas expressed. (J= to L=) Presence of NeurWT,
Neur?NHR2, and NeurG430E(green).
Liu et al.
mcb.asm.orgMolecular and Cellular Biology
WT control (Fig. 7G, compare IP lanes 3 and 4 on the anti-FLAG
blot). Of note, the difference in binding is unlikely to be due to
differences in protein expression levels or immunoprecipitation
efficiencies, since we observed comparable levels of wild-type and
mutant proteins in both the input (Fig. 7G, input lanes 3 and 4)
and immunoprecipitated baits (Fig. 7G, IP lanes 3 and 4 on the
anti-V5 blot). Furthermore, when we repeated the co-IP experi-
ments with Kc cells cotransfected with NeurG430E-V5and
NeurG430E-FLAGfollowed by anti-V5 immunoprecipitation, we
found that the oligomerization of NeurG430Ewas compromised
(Fig. 7G, lane 9).
We also examined whether the presence of the G430E muta-
tion in the NHR1 deletion protein (Neur?NHR1G430E-FLAG)
affected the binding to Neur lacking the NHR2 domain
(Neur?NHR2-V5). As before, we observed similar levels of input
(Fig. 7B, input lanes 5 and 11) and immunoprecipitated V5-
tagged baits (Fig. 7B, IP lanes 5 and 11), but the level of coim-
munoprecipitated FLAG-tagged Neur?NHR1G430E
creased compared to that of Neur?NHR1(Fig. 7B, compare IP
lane 11 to lane 5 on the anti-FLAG blot), suggesting that the
G430E mutation allows Neur?NHR1G430Eto bind more effi-
ciently with Neur?NHR2than with Neur?NHR1. Taken together,
our data suggest that Neur forms oligomers via the two NHR
ing RING domain with the interstitial regions could still
direct Neur oligomerization. Nevertheless, the Gly430 residue
FIG 6 Asymmetric localization of Neur lacking a PIP-binding motif depends on endogenous Neur. (A) Neur (red) localizes asymmetrically in an anterior
V5-tagged Neur-PA (F) localized asymmetrically. In contrast, Neur-PC (G) and Neur-5Q (H) were not asymmetric.
NHR2 Domain Regulates Neur Activity
December 2012 Volume 32 Number 24mcb.asm.org 4941
in NHR2 plays an important role in this oligomerization
signaling by regulating the endocytosis of Notch ligands. It con-
or plants. In the Drosophila proteome, besides Neuralized, there
are two other NHR-containing proteins, CG3894 and Bluestreak.
In mammals, proteins containing NHR domains (also known as
NEUZ) include the ?-catenin regulator OzzE3 (40) and lung-in-
ducible Neuralized-related C3HC4 RING protein (LINCR) (21).
Recent studies reported that the human homologue of Bluestreak
serves to localize to the centrosome (1, 37). Although the general
and most proteins contain two to six NHR domains. The signifi-
cance of having more than one NHR domain in one protein is to
yet be determined.
Here we have investigated the role of the highly conserved
FIG 7 Neuralized forms oligomers in Kc cells. (A) Schematic of tagged wild-type and deletion mutants of Neur for coimmunoprecipitation. Kc cells were
cotransfected with differently tagged Neur protein variants, followed by an anti-V5 immunoprecipitation. (B and C) In each co-IP, pairs of Neur variants were
tested for oligomerization. The deletion of either NHR domain or both NHR domains did not affect Neur oligomerization. FLAG-tagged deletion mutant
proteins of both NHR domains are indicated by the two-headed arrow (C) (*, nonspecific bands). Yeast two-hybrid analysis revealed that Neur interacts with
“?” indicates a weak interaction, and “?” indicates no observed interaction. (F) The FLAG-tagged NHR2 domain was pulled down by the V5-tagged NHR1
domain in co-IP experiments. The input contains 1/20 of the lysate used for immunoprecipitation. (G) In the immunoprecipitation blot, FLAG-NeurWTwas
Liu et al.
mcb.asm.orgMolecular and Cellular Biology
alone mediates the interaction between Neur and both Delta and
function, and while it is not required for the interaction with
nation activity and ligand endocytosis.
The NHR1 domain mediates the interaction between Neur
and the Notch ligands Delta and Serrate. We have previously
shown that the NHR1 domain of Neur is both necessary and suf-
ficient for the interaction with the Notch ligand Dl (9). Specifi-
to bind to Serrate, however, was unknown. In fact, in vitro studies
ized-like 1 (Neurl1) was sufficient to bind to Jagged1, the mouse
orthologue of Serrate (26). In contrast, we find that the NHR2
that the interaction is mediated entirely by the NHR1 domain.
Other studies reported previously that the motifs on Dl and Ser
that mediate the interaction with Neur are conserved (11, 16). In
comparisons of protein sequences, Jagged1 and Serrate share
40.7% similarity overall, while the overall similarity between Jag-
ged1 and Dl is 33.8% (39). The NHR1 and NHR2 domains from
Drosophila Neuralized have the same degree of amino acid simi-
larity with the mouse Neurl1 NHR2 domain (33%). Since there is
no clear correlation between protein sequence similarity and the
ability of either the NHR1 or the NHR2 domain to interact with
ing with ligands is likely to be species dependent. We found that
the Neur-Ser interaction is abrogated by the G167E mutation in
the NHR1 domain. Given that the NeurG167Emutant still retains
ubiquitination activity, it is unlikely to affect overall protein fold-
ing. A previously reported structural analysis of the Drosophila
NHR1 domain suggested that Gly167 resides in a hydrophobic
core and that the Gly167 mutation presumably destabilizes the
surrounding microenvironment (19). Therefore, the Gly167 mu-
the core, thus abolishing binding to ligands.
main in the regulation of Neur activity and Delta trafficking.
function in vivo. NeurG430Efails to rescue neur mutant embryos,
while Neur?NHR2has some residual activity, which suggests that
they affect different aspects of Neur function. The expression of
NeurG430in a heterozygous background, which does not have a
in the percentage of neurogenic embryos, suggesting that
NeurG430Ehas a negative effect on Neur function. In contrast,
Neur?NHR2overexpression did not have any effect on heterozy-
gous embryos, suggesting that it behaves as a loss-of-function al-
and Neur?NHR2behave differently, they both localize to the
plasma membrane in the presence of Delta both in vitro and in
vivo, and they are both capable of binding to the Notch ligands
Delta and Serrate. However, both mutant proteins affect the ex-
tent of Dl internalization to various degrees. NeurG430Eexhibits
severely compromised ubiquitination activity and is no longer
capable of inducing Delta internalization. Neur?NHR2, on the
other hand, retains ubiquitination activity but is much less effi-
cells. The precise mechanism by which Neur?NHR2affects Delta
endocytosis is unclear.
One possibility is that the NHR2 domain is required for Neur
oligomerization. We have shown that Neur forms NHR domain-
mediated oligomers by coimmunoprecipitation experiments.
Therefore, the deletion of the NHR2 domain (Neur?NHR2) may
simply reduce the oligomerization potential of Neur, leading to a
decrease in ligand endocytosis. In contrast, the point mutation
(NeurG430) might disrupt the overall structure of the NHR2 do-
main, preventing oligomerization and resulting in a protein that
has no ubiquitination activity and therefore can no longer inter-
which show that ?NHR2 does not prevent Neur oligomerization
and that Neur oligomerization still occurs in the absence of any
NHR domains, suggesting that while the NHR domains may play
a role in oligomerization, they are not necessary for this process.
Our data also show that although the G430E mutant loses ubiq-
tion and the G430E mutation retains ubiquitination activity,
which argues that G430E does not affect the overall folding of the
Another possibility is that the NHR1 and NHR2 domains ini-
resolved for ubiquitination to promote ligand internalization
(Fig. 8B). The G430E mutation may lock Neur into an intramo-
lecular conformation through an NHR1-NHR2 interaction, such
oligomers and has no ubiquitination activity as a consequence of
dysfunctional oligomerization (Fig. 8C). This model, in contrast
to the former one, is supported by our data, which demonstrate
that the G430E mutant no longer forms oligomers and has no
ubiquitination activity. Furthermore, when the NHR1 domain
was removed from the G430E mutant (Neur?NHR1G430E), the
ubiquitination activity was restored, suggesting that the intramo-
lecular loop can no longer form, whereas intermolecular interac-
tions between NHR1 and NHR2 domains can occur. It may also
explain the negative effect of NeurG430Eon NeurWT: although
NeurG430Ehas a reduced ability to bind WT Neur, it is still be able
to sequester some portion of NeurWTinto a nonfunctional inter-
molecular oligomer that can no longer ubiquitinate targets. In
contrast, the deletion of the NHR2 domain would prevent intra-
molecular interactions but would be expected to have a reduced
ability to form productive oligomers, leading to a defect in ligand
internalization. Whether the NHR2 domain has additional roles
as oligomers, and they multimerize in different ways: some form
heterodimers, such as Mdm2-MdmX (38), and some can form
homo-oligomers, such as TRAF (55). The functional significance
of RING E3 oligomerization is poorly defined. One previously
proposed model is that the oligomerization of E3 ligases may
functionally resemble the dimerization of receptor tyrosine ki-
nases in such a way that autoubiquitination yields a mark that
NHR2 Domain Regulates Neur Activity
December 2012 Volume 32 Number 24mcb.asm.org 4943
tent with this idea, we did see ubiquitination in anti-Neur IPs
when Dl was not present, consistent with the idea that Neur may
be autoubiquitinated (12). Whether this autoubiquitination can
initiate a cascade of further downstream ubiquitination events
remains to be determined. It is possible that oligomerization is
mediated via autoubiquitination and the interaction of ubiquiti-
nated Neur with itself through a ubiquitin-binding motif (UIM).
If so, then the NHR1-NHR2 interaction could also function to
keep Neur in an inactive state by occluding the putative UIM.
posed for other endocytic proteins (45).
In summary, we have shown that NHR domains are protein-
protein interaction modules that are required for many aspects of
Neur function. The NHR1 domain mediates the interaction be-
to regulate Neur activity by affecting its ability to form oligomers
and/or interact with proteins required for the endocytosis of
Notch ligands. Interestingly, NHR domains have been identified
in several other proteins that are conserved between flies and hu-
mans. Whether Neur can form heterodimers with these other
NHR-containing proteins and whether these heterodimers play a
to be determined.
We thank S. Egan and F. Sicheri as well as members of the Boulianne
laboratory for helpful discussions and critical comments on the manu-
DSHB, and the Bloomington Stock Center for reagents.
is supported through a studentship, in part, by the Research Training
Competition (Restracomp) Hospital for Sick Children Foundation Stu-
dent Scholarship Program. J.M.B. is the recipient of a CIHR graduate
scholarship. G.L.B. holds a tier 1 Canada research chair in molecular and
developmental neurobiology. This work was funded by a grant from the
Natural Sciences and Engineering Council of Canada to G.L.B.
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