From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, April 11, 2001, and in revised form, May 4, 2001
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ABSTRACT |
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Rapsyn, a peripheral membrane protein of skeletal
muscle, is necessary for the formation of the highly organized
structure of the vertebrate neuromuscular junction. For mice lacking
rapsyn, there is a failure of postsynaptic specialization characterized by an absence of nicotinic acetylcholine receptors (nAChRs) and other
integral and peripheral membrane proteins such as Rapsyn, a 43-kDa peripheral membrane protein expressed in skeletal
muscle, plays a critical role in organizing the structure of the
nicotinic postsynaptic membrane (for review, see Ref. 1). Rapsyn is
colocalized with nicotinic acetylcholine receptors
(nAChRs)1 in the postsynaptic
membrane from the earliest stages of innervation (2), and this precise
colocalization is a characteristic feature of the adult neuromuscular
junction (3). In rapsyn( Although the three-dimensional structure of rapsyn is not known, its
primary structure suggests the presence of distinct structural domains:
a myristoylated amino terminus (14), as many as eight tetratricopeptide
repeats (TPRs) within rapsyn6-319 (15), a coiled-coil
domain (rapsyn298-331) (16), a cysteine-rich domain
(rapsyn363-402) predicted to be a RING-H2 domain (17), and
a consensus sequence for phosphorylation by protein kinases A and C
(rapsyn403-406). Previously, in studies of rapsyn and
rapsyn mutants expressed transiently in 293T cells along with nAChRs,
we identified the rapsyn structural domains involved in its membrane
targeting, self-association, and nAChR-clustering properties (16, 18).
Rapsyn plasma membrane targeting requires amino-terminal fatty
acylation, whereas rapsyn self-association requires at least two TPRs.
Rapsyn clustering of nAChRs depends upon the presence of the putative
coiled-coil domain within rapsyn, and a construct containing as few as
3 TPRs and the coiled-coil domain was sufficient for nAChR clustering.
Deletion of the rapsyn coiled-coil domain prevented rapsyn clustering
of nAChRs. These studies clearly established that neither the rapsyn
RING-H2 domain nor the adjacent phosphorylation sites were necessary
for membrane targeting, self-association, or nAChR clustering.
We report here a further analysis of rapsyn functional domains using a
yeast two-hybrid assay as well as additional studies of rapsyn mutants
expressed in 293T cells. To test for interactions at the plasma
membrane between rapsyn structural domains and rapsyn-associated proteins, we used a yeast two-hybrid assay (19) that depends upon the
recruitment of human Son of Sevenless (hSos) to the plasma membrane to
rescue Ras-mediated signal transduction. We tested for interactions
between rapsyn domains fused to hSos and candidate protein domains
anchored in the plasma membrane by fusion with the
N-myristoylated v-Src amino terminus. With this assay we
looked for interactions within rapsyn and for interactions of the
rapsyn domains with the membrane-anchored cytoplasmic domain of the
nAChR Materials
All restriction enzymes were purchased from New England Biolabs.
T4 DNA ligase and oligonucleotides were obtained from Life Technologies, Inc. Polymerase chain reactions (PCR) were carried out in
100 µl using 20 ng of the templates described below, 50-100 pmol of
each primer, 250 µM each dNTP, and 5 units of
Pfu DNA polymerase (Stratagene) for 30 cycles at 95 °C
for 1 min, 55 °C for 1 min, and 72 °C for 1 min for 30 cycles.
All constructs were tested both by restriction enzyme analysis and by
sequencing across the full length of the inserted fragments. Yeast
cdc25H Saccharomyces cerevisiae as well as the vectors pSos
and pMyr were purchased from Stratagene. The vector p5'Sos (19) was
provided by Dr A. Aronheim (Technion, Haifa, Israel). The plasmids
pCDNA3-dystroglycan (mouse) and pCDNA3-dystroglycan (rabbit)
were provided by Drs. S. Carbonetto (McGill University) and K. P. Campbell (University of Iowa), respectively.
Plasmid Construction
Sos-Rapsyn Constructs--
Human Sos was fused at its COOH
terminus to full-length rapsyn (Sos-rapsyn1-412), to the
rapsyn RING-H2 domain (Sos-rapsyn361-412), and to the
rapsyn coiled-coil domain (Sos-rapsyn298-333). For
Sos-rapsyn1-412 the primers
GCCACCATGCGGCCGCACCAGACAA and GGAAACCCAAGGCGGCCGCTGAAG (nucleotides in
bold are mismatched to create the underlined NotI site) were
used along with the template pGL-rapsyn1-412 (16) in a PCR
to amplify rapsyn amino acids 1-412. The PCR product was digested with
NotI, and the fragment containing the rapsyn sequence was
cloned into pSos vector linearized with NotI. For
Sos-rapsyn361-412, the primers
GTGGAGGAGACTGGGATCCACTGCGGCCTC and AGATGGCCGCTGAGCTCACACAAAGCCCGG
(nucleotides in bold are mismatched to create the underlined
BamHI and SacI sites) were used with the same
template to amplify rapsyn amino acids 361-412. For
Sos-rapsyn298-333 the primers
CAAGTGCTGGATCCCCCGGAAGG and
GCGGTAGATGAGCTCACTCAGG also containing
BamHI and SacI sites were used along with the
template to amplify rapsyn amino acids 298-333. The PCR products were
digested with BamHI and SacI, and the fragments
containing the rapsyn sequences were cloned into the pSos vector.
Rapsyn-Sos Constructs--
Rapsyn domains lacking the rapsyn
N-myristoylation consensus sequence were fused at the
NH2 terminus of human Sos in rapsyn1-360-Sos, which lacks the RING-H2 domain, and in rapsyn1-287-Sos, encoding the first seven TPRs of rapsyn. For
rapsyn1-360-Sos the primers
GATCCCAAGCTTACCATGGAGATCATGGCGCAGGACCAGAC and
GAGGATCCTCACCCGGGAGTCTCCTCCAGCACTC (underlined are the
HindIII and XmaI sites) were used along with the
template pGL-rapsyn1-412G2A-GFP (which encodes a rapsyn Gly2Ala mutation (16)) in a PCR to amplify rapsyn amino acids 1-360.
The PCR product was cloned into pGEM T-easy (Promega), the plasmid was
digested with HindIII and XmaI, and the fragment containing rapsyn was subcloned into the p5'Sos vector. To make rapsyn1-287-Sos, rapsyn1-360-Sos was digested
with PmlI and SmaI, and the vector fragment was religated.
Myristoylated Rapsyn Constructs--
Rapsyn domains were
targeted to the plasma membrane by fusion with the amino terminus of
v-Src (MGSSKSKPKDPSQRRSREF), which contains the
NH2-terminal myristoylation and membrane-targeting sequences. For Myr-rapsyn5-412 the cDNA encoding
full-length rapsyn1-412 was excised with EcoRI
from the pSLAN-rapsyn vector2
and then inserted into the pMyr vector linearized by EcoRI.
Due to the restriction site used in the pSLAN-rapsyn vector, the first four amino acids of rapsyn (MGQD) were replaced by the sequence KRPH.
Myr-rapsyn5-412 was digested with NotI and
SacI, and the vector fragment was blunted and then religated
to create Myr-rapsyn360-412 containing the rapsyn RING-H2
domain. Myr-rapsyn5-412 was digested with SacI
and XhoI, and the vector fragment was blunted and then
religated to form Myr-rapsyn5-360, which lacks the RING-H2 domain.
Myr-nAChR Dystroglycan776-893-Sos--
This construct encodes
the cytoplasmic domain of Myristoylated pGL-rapsyn1-90+351-412,
pGL-rapsyn1-254+351-412, and
pGL-rapsyn1-287+351-412--
pGL-rapsyn (16) was
digested with either BstUI (for 1-90; includes
N-Myr-TPRs1+2), EcoRV (for 1-254; includes N-Myr-TPRs1-6), or PmlI (for 1-287; includes N-Myr-TPRs1-7) and
NotI. The vector fragment was isolated in each case and
ligated with the BsaAI-NotI fragment of rapsyn,
which encodes rapsyn 351-411 containing the RING-H2 domain.
Yeast Transformation and Interaction Testing
All media were made as described (19) with reagents from Bio
101. The yeast strain cdc25H was cotransfected with 1 µg
of each plasmid using the alkali-cation yeast kit from Bio 101. Cells were resuspended in 200 µl of SOC medium and plated on glucose minimal medium lacking leucine and uracil. To test for protein interactions, yeast cells were picked and spotted onto plates containing glucose or galactose and incubated for 4 days at 25 or
37 °C. Plates were scanned to obtain digitized images. For colonies
where no growth on galactose was seen after 4 days at 37 °C, no
growth was observed after incubation for several days longer.
Expression of Rapsyn, Rapsyn Mutants, and Dystroglycan in 293T
Cells--
Transfection of 293T cells by the calcium phosphate method,
cell staining, and immunofluorescence experiments were done as described (18), except that after fixation, the cells were usually permeabilized with 1% Triton X-100 for 10 min and blocked with blocking buffer for 30 min before incubation with the primary antibody.
Cells were visualized using a Nikon Eclipse E800 epifluorescence microscope with a Nikon 100X Plan Fluor objective (NA1.3). Images were
acquired with a Micromax CH250 CCD camera (Princeton Instruments) using
MetaMorph software.
Rapsyn or its fragments were visualized by the binding of either
anti-rapsyn (1-16), an affinity-purified rabbit polyclonal antibody
directed against a synthetic peptide corresponding to the
NH2-terminal 16 amino acids of rapsyn (non-myristoylated) (at 2 µg/ml), or mouse monoclonal mAb 22F10 (20) (1:1000 dilution of
ascites), followed by the appropriate ALEXATM488-conjugated
secondary antibody (1:300 dilution, Molecular Probes). mAb 22F10 was
prepared by using Torpedo rapsyn as immunogen. Based upon
epitope-mapping studies mAb 22F10 recognizes N-myristoylated rapsyn amino acids 1-11 with >1000-fold greater affinity than the
non-myristoylated
peptide.3
The distribution of dystroglycan was visualized by binding to one of
the antibodies listed below followed by the appropriate ALEXATM594- or Cy3-conjugated secondary antibody. 1)
Affinity-purified sheep polyclonal FP-B directed against a bacterial
expressed fusion protein encoding amino acids 367-863 of rabbit
Quantification of Cells with Rapsyn or Dystroglycan
Clusters--
For experiments involving expression of rapsyn or its
mutants and dystroglycan in 293T cells, quantification of dystroglycan clusters relative to rapsyn clusters was done as follows. In each experiment, 100 cells positive for both rapsyn and dystroglycan expression were identified. For these we quantified the number of cells
with dystroglycan that colocalized with rapsyn clusters. Each
experiment was repeated at least three times, cells were scored as
above, and the results are presented as the % of cells with (or
without) clustered dystroglycan.
To provide a further analysis of the properties of the rapsyn
structural domains, we wanted to identify the binding properties of the
individual domains, and we were particularly interested in identifying
proteins interacting with the RING-H2 domain. However, we were
concerned that RING domains often have a strong tendency to aggregate
(24-26), and some self-activate when tested in transcription factor
yeast two-hybrid assays (27). As an alternative, we tested for the
interactions of rapsyn domains with potential partners by use of a
yeast two-hybrid assay that identifies protein interactions at the
plasma membrane (19). The assay takes advantage of a yeast strain that
is temperature-sensitive for the Ras exchange factor cdc25. At the
permissive temperature of 25 °C, the temperature-sensitive mutant
cdc25H is functional, and the yeast grow well, but at
37 °C, cdc25H is nonfunctional, and the yeast fail to
grow. The expression of hSos, the mammalian homologue of cdc25, is able
to complement the cdc25 mutation only if it can be localized to the
plasma membrane. hSos membrane localization can be accomplished if it
is expressed as a hybrid protein fused to a bait protein that interacts
with a partner which is localized at the plasma membrane. Localization of the partner to the plasma membrane is achieved by fusing it to
the myristoylated amino terminus of v-Src. The expression of the
partner is driven by the Gal1 promoter, which is induced by galactose.
When the bait and the partner are both expressed and interact, the
Ras-signaling pathway is rescued, allowing the growth of yeast at
37 °C on plates containing galactose.
Interaction Partners for Rapsyn Structural Domains--
Fig.
1 presents the results obtained using
individual domains of rapsyn fused with hSos as bait and full-length
rapsyn or the cytoplasmic domain of the nAChR
Sos-rapsyn298-333 containing only the rapsyn coiled-coil
domain interacted with Myr-nAChR
Sos-rapsyn361-412, interacted with
Myr-dystroglycan778-893, containing the full COOH-terminal
cytoplasmic domain of Mapping the Region of Rapsyn Clustering of Dystroglycan in 293T Cells--
When
expressed in QT-6 fibroblasts, dystroglycan is distributed uniformly at
the cell surface, but when co-expressed with rapsyn, dystroglycan is
colocalized with clustered rapsyn (11). We wanted to identify the
rapsyn domains required to recruit dystroglycan to rapsyn clusters. We
initially planned to express rapsyn-GFP constructs (16, 18) with
dystroglycan in 293T cells and visualize the expressed rapsyn domains
by the fluorescence of GFP and dystroglycan by the binding of
appropriate antibodies. We were surprised to find, however, that
rapsyn-GFP was not able to cluster dystroglycan (data not shown) and
that even with full-length rapsyn, the clustering of dystroglycan could
be seen only with one of the five dystroglycan antibodies tested, the
FP-B polyclonal antibody that had been used in previous studies of
dystroglycan clustering (11). For cells coexpressing rapsyn and
dystroglycan,
Dystroglycan clustering by rapsyn was readily apparent in >95% of
cells expressing both proteins when the transfected dystroglycan was
detected using the FP-B antibody under permeabilized conditions (Fig.
3, i (rapsyn) and j (dystroglycan)). For 293T
cells transfected with dystroglycan in the absence of rapsyn,
dystroglycan was distributed diffusely at the cell surface as seen with
the other
In additional experiments, when rapsyn and dystroglycan were
coexpressed in QT-6 fibroblasts and dystroglycan was visualized by the
FP-B antibody, it was clustered with rapsyn. When dystroglycan was
visualized by the other antibodies, the staining was seen to be
distributed diffusely (data not shown). In the following experiments to
identify the rapsyn domains involved in dystroglycan clustering, we
used the FP-B antibody to visualize transfected dystroglycan.
Rapsyn Domains Necessary for Dystroglycan Clustering--
As a
first test of the requirement of the RING-H2 domain for the clustering
of dystroglycan, we attempted to coexpress dystroglycan with
rapsyn1-360, lacking the RING-H2 domain. Unfortunately, in
contrast to rapsyn1-412 or rapsyn1-360-GFP
(16), based on immunofluorescence there was no evidence of expression of rapsyn1-360 (or clustering of dystroglycan). To
identify the rapsyn domains required in conjunction with the RING-H2
domain for dystroglycan clustering, we expressed three rapsyn
constructs with varying numbers of TPRs fused to the RING-H2 domain
(Fig. 4a).
Rapsyn1-287+351-412, consisting of seven TPRs fused to
the RING-H2 domain but lacking the coiled-coil domain, formed clusters
similar to wild-type rapsyn and clustered dystroglycan in all the cells
expressing both proteins (Fig. 4, b and c), as did rapsyn1-254+351-412, with six TPRs and the RING-H2
domain (Fig. 4, d and e).
Rapsyn1-90+351-412, with only TPRs 1 and 2 and the
RING-H2 domain, also formed distinct clusters at the plasma membrane
and clustered dystroglycan in all cells expressing both proteins (Fig.
4, f and g). No clustering of nAChRs was seen for
any of these three rapsyn constructs lacking the coiled-coil domain
(Fig. 4, h-m).
The results presented here extend our understanding of the
functional roles of the rapsyn TPR and coiled-coil domains and identify
for the first time a binding partner, As an alternative approach to studying the functional properties of
individual rapsyn domains, we used the yeast two-hybrid assay based on
hSos recruitment (19) to test for interactions between rapsyn domains
and potential interacting domains from rapsyn, the nAChR The Rapsyn RING-H2 Domain and Dystroglycan--
Biochemical
studies (6) provided the first evidence, based upon a gel "overlay
assay," that dystroglycan787-819, the membrane proximal
portion of the cytoplasmic domain, can bind to rapsyn. Our results now
establish that it is the RING-H2 domain of rapsyn that interacts with
that region of The Stability of Expressed Rapsyn Domains--
In the hSos
recruitment assay, one of the interacting proteins is anchored at the
plasma membrane by fusion with the NH2-terminal v-Src
membrane-targeting sequence, and the other protein can be expressed
either as an NH2-terminal or COOH-terminal hSos fusion protein. In our yeast assay we did not systematically test both fusion
orientations. Rather, we were guided by previous results obtained in
the 293T expression system (16). Thus, we expressed rapsyn1-287-Sos and rapsyn1-360-Sos because
those rapsyn fragments, when fused at their COOH termini to GFP,
expressed well. Interestingly, when we coexpressed
Sos-rapsyn1-360 with Myr-rapsyn5-412, there
was no yeast growth under selective conditions, and based upon
immunofluorescence, we found no evidence of the expression of
rapsyn1-360 protein in the 293T assay. In contrast,
Sos-rapsyn1-360-GFP was expressed and interacted with
Myr-rapsyn5-412. Thus, it appears that expressed rapsyn COOH-terminal truncation mutants can be unstable if the normal COOH-terminal RING-H2 domain is removed, but the expressed proteins can
be stabilized by either GFP or hSos at the COOH terminus. The rapsyn
coiled-coil and RING-H2 domains were readily expressed when fused at
the COOH terminus of hSos, and the coiled coil domain was also
expressed when fused at the COOH terminus of the v-Src membrane
targeting sequence (not shown).
Rapsyn Clustering of Dystroglycan in 293T Cells--
We were
surprised that in the 293T cell assay, dystroglycan clustering by
rapsyn was seen only when dystroglycan was visualized by the FP-B
antibody and not when visualized by two monoclonals specific for
Mapping the Interactions between Rapsyn RING-H2 Domain and
RING zinc finger domains mediate protein-protein interactions involved
in the formation of large molecular scaffolds (25, 26). Some RING
domains self-associate or bind to other RING domains, whereas at least
three other RING binding domains recognize a proline-rich sequence
defined by PXBXPJXP, where B and J are Leu/Val and Ala/Ser, respectively. In the complex between the c-Cbl
proto-oncogene, a RING domain ubiquitin-protein ligase, and a ubiquitin
conjugating enzyme, side chains on one of the surfaces of the c-Cbl
RING domain (including cysteines also involved in zinc coordination)
interact with amino acids in loops on the surface of the conjugating
enzyme, whereas a second surface of the RING domain interacts with
another domain within the ligase (29). Our results establish that the
rapsyn RING-H2 domain is clearly not involved in self-association and
that it binds to a protein lacking a RING domain. Although the membrane
proximal Rapsyn RING-H2 Domain, Dystroglycan, and nAChR Clustering--
Our
studies demonstrate that the rapsyn-RING-H2 domain interacts with the
Several studies provided indirect evidence that the rapsyn RING-H2
domain might be involved in interactions with -dystroglycan and
utrophin. Dystroglycan is necessary for the formation of the mature
neuromuscular junction and has been shown to interact directly with
rapsyn. Previous studies with rapsyn fragments and mutants, expressed
in 293T cells along with nAChRs, establish that the rapsyn
tetratricopeptide repeat (TPR) domain is involved in self-association and its coiled-coil domain is necessary for nAChR clustering. The
function of the rapsyn RING-H2 domain, which is not necessary for
rapsyn self-association or nAChR clustering, is unknown. To further
characterize these domains, we have used a yeast two-hybrid assay to
test for interactions at the plasma membrane between rapsyn domains and
a nAChR
-subunit fragment, the
-dystroglycan cytoplasmic domain,
or rapsyn domains. The rapsyn coiled-coil domain interacts with the
nAChR
-subunit cytoplasmic domain, but not with itself, other rapsyn
domains, or
-dystroglycan. The RING-H2 domain interacts only with
the
-dystroglycan cytoplasmic domain. Furthermore, when expressed in
293T cells, a rapsyn construct containing as few as two TPRs and the
RING-H2 domain self-associates and clusters dystroglycan, but not
nAChRs. These results emphasize the modular character of the rapsyn
structural domains.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mutant mice, which die at birth because
of a failure of neuromuscular transmission, nAChRs are expressed by the
subsynaptic nuclei and targeted to the postsynaptic plasma membrane,
but they are not clustered (4). These mice are characterized by a
general disorganization of the postsynaptic membrane, as utrophin (a
cytoskeletal protein that is normally colocalized with nAChRs) and
-dystroglycan (the transmembrane protein that binds utrophin) are no
longer enriched in the postsynaptic membrane. Biochemical studies using
nAChR-rich membranes from Torpedo electric organ indicate
that rapsyn can be cross-linked to the nAChR
-subunit (5) and bind
to the cytoplasmic domain of
-dystroglycan (6). When expressed in non-muscle cells, rapsyn forms membrane-associated clusters and recruits nAChRs to these clusters (7, 8). The nAChR domain necessary
for this interaction has been located within a region of primary
structure between the M3 and M4 hydrophobic segments, which is known to
be exposed at the cytoplasmic aspect of the nAChR (9, 10). In
non-muscle cells, rapsyn can also cluster
-dystroglycan (11). Thus,
rapsyn may function as a direct link between nAChRs and the
dystrophin/utrophin-associated glycoprotein complex (12, 13) that
extends from the extracellular matrix to the cytoskeleton.
-subunit or of
-dystroglycan.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
336-469--
This construct encodes
amino acids of the nAChR
subunit between the M3 and M4 hydrophobic
segments fused to the v-Src amino terminus. The
primers
TCAACCCTGCATGAATTCTCACCCCACACC and
GAATGTCCACAGCTCGAGACGGTCCACAAC (underlined are the
BamHI and XhoI sites) were used to amplify nAChR
subunit amino acid sequence 336-469. The PCR product was cloned
into pGEM T-easy, the plasmid was digested with BamHI and XhoI, and the fragment containing the nAChR
-subunit
sequence was subcloned into the pMyr vector.
-dystroglycan fused at the amino
terminus of hSOS. The primers
GATCCCAAGCTTACCATGGAGATCTATCGC and
GAGGATCCTCACCCGGGAGGGGGAACATACGGAGG (underlined are the
HindIII and XmaI sites) were used along with the
template pCDNA3-dystroglycan (mouse) in a PCR to amplify
-dystroglycan amino acids 776-893. The PCR product was cloned into
pGEM T-easy, the plasmid was digested with HindIII and
XmaI, and the fragment containing
-dystroglycan was
subcloned into the p5'Sos vector.
-Dystroglycan Constructs--
-Dystroglycan
domains were targeted to the plasma membrane by the v-Src
NH2-terminal membrane-targeting sequence.
Myr-dystroglycan778-893 encodes the complete cytoplasmic
domain, whereas Myr-dystroglycan778-814 encodes the
proximal, and Myr-dystroglycan813-893 encodes the distal
cytoplasmic domain. For Myr-dystroglycan778-893, primers U
(AGCTAAGCTTATCCGCAAGATGGGGAAGGG) and B
(TCGACCCGGGAAGGGGAACATACGG) were used to amplify
dystroglycan amino acids 778-893 (the HindIII and
XmaI sites are underlined). For
Myr-dystroglycan778-814 we used the primer U and the
primer TCGACCCGGGCGGCATGCTGGAAGAGGG (the XmaI
site is underlined) to amplify dystroglycan amino acids 778-814. For
Myr
-dystroglycan813-893 we used the primer CGTAAGCTTTTCCAGCAATGGGGCTCATCTTG (the HindIII
site is underlined) and primer B to amplify dystroglycan amino acids
813-893. The PCR products were cloned in pGEM T-easy, the
resulting plasmids were digested with HindIII and
XmaI, and the fragments containing
-dystroglycan were subcloned into pMyr vector.
/
-dystroglycan (Ref. 21; a gift from Dr. K. P. Campbell) was
used at a dilution of 1:10 to visualize transfected dystroglycan in
293T cells that were fixed and permeabilized. No fluorescence signal
was seen in transfected cells without permeabilization or in
non-transfected cells. 2) Mouse monoclonal NCL-b-DG (1:100 dilution of
hybridoma supernatant, Novocastra Laboratories) was raised against the
COOH-terminal 15 amino acids of human
-dystroglycan. 3)
Affinity-purified rabbit polyclonal antibody P-20 (4 µg/ml, a gift
from Dr. T. Petrucci, Instituto Superiore di Sanita, Rome, Italy) was
directed against the COOH-terminal 20 amino acids of
-dystroglycan
(22). 4) Anti-
-dystroglycan mouse monoclonal mAb 1B7 (1:100 dilution
of ascites, a gift from Dr. S. Carbonetto) was raised against a
33-amino acid synthetic peptide corresponding to mouse dystroglycan
amino acids 572-604 at the
-dystroglycan COOH terminus (23). 5)
Anti-
-dystroglycan mouse monoclonal antibody IIH6 (Ref. 21; IgM, a
gift from Dr. K. P. Campbell) was used undiluted or at a dilution
1:5 of hybridoma supernatant. Alexa594-conjugated secondary antibodies
(Molecular Probes) were used at a 1:300 dilution. Cy3-conjugated goat
anti-mouse IgM (1:200 dilution, Chemicon) was used to detect mAb IIH6.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit or
-dystroglycan as target. As negative controls each construct tested
was cotransfected with either a myristoylated transcription factor
(Myr-MafB) or hSos alone, and no growth at 37 °C was seen with any
construct. Based upon the growth seen under selective conditions,
Myr-rapsyn5-412 interacted with
Sos-rapsyn1-412, rapsyn1-360-Sos, and
rapsyn1-287-Sos but not with
Sos-rapsyn298-333 or Sos-rapsyn361-412 (Fig.
1, third row). These data indicate that the
rapsyn TPR domain (rapsyn1-287) can associate with full-length rapsyn, whereas the coiled-coil domain or the RING-H2 domain cannot. Although the RING-H2 and coiled-coil domains did not
interact with rapsyn, they were each able to interact with domains of
other proteins (see below).
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Fig. 1.
Interactions between rapsyn domains and
rapsyn, nAChR -subunit, and
-dystroglycan (DG).
Complementation of the cdc25H mutation through the
interaction of rapsyn domains with potential binding partners is shown.
Temperature-sensitive cdc25H yeast cells were cotransfected
with combinations of the indicated rapsyn domains fused to hSOS
(x axis) and N-myristoylated (N-MYR),
membrane-targeted potential interaction partners (y axis).
Colonies were picked and replica-plated onto plates containing glucose
or galactose and incubated at either 25 or 37 °C as indicated.
Plasmids encoding hSos alone or the transcription factor MafB fused
with the v-Src membrane-targeting signal were used as negative
controls. A schematic diagram of the structural domains of rapsyn is
included at the top.
336-469, the
membrane-targeted
-subunit cytoplasmic domain. This interaction of
the nAChR cytoplasmic domain with the rapsyn coiled-coil domain was
very selective, because it also only interacted with rapsyn constructs
containing the coiled-coil domain (Sos-rapsyn1-412 and
rapsyn1-360-Sos) but not with rapsyn1-287-Sos
(comprising TPR 1-7) or with Sos-rapsyn361-412,
containing the RING-H2 domain (Fig. 1, second row).
-dystroglycan, and
Myr-dystroglycan778-893 interacted with full-length rapsyn
(Sos-rapsyn1-412) but not with rapsyn constructs lacking
the RING-H2 domain (rapsyn1-360-Sos, rapsyn1-287-Sos, or Sos-rapsyn298-333) (Fig.
1, first row). These results indicate that the rapsyn
RING-H2 domain is directly involved in the interaction with the
cytoplasmic domain of
-dystroglycan and that neither the TPR domain
nor the coiled-coil domain of rapsyn can bind to the
-dystroglycan
cytoplasmic domain. To confirm these results, we determined that not
only can
-dystroglycan recruit rapsyn or its RING-H2 domain to the
plasma membrane but that membrane associated domains of rapsyn
containing the RING-H2 domain can also recruit the
-dystroglycan
cytoplasmic domain to the plasma membrane (Fig.
2A). Dystroglycan776-893-Sos was able to interact only with the RING-H2 domain
(Myr-rapsyn360-412) or full-length rapsyn
(Myr-rapsyn5-412) but not with
Myr-rapsyn5-360.
View larger version (31K):
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Fig. 2.
Mapping the
-dystroglycan domain that binds to the rapsyn
RING-H2 domain. A, yeast cells cotransfected with the
cytoplasmic domain of
-dystroglycan in the p5'Sos vector and the
indicated rapsyn constructs in the pMyr vector or with Myr-MafB as a
negative control were spotted on plates containing galactose and
incubated for 4 days at 37 °C. In a parallel experiment, no growth
was seen at 37 °C when the cotransfected yeast were spotted on
plates containing glucose (not shown). B, plasmids encoding
N-myristoylated (Myr) membrane proximal
(Myr-DG778-814) or membrane distal
(Myr-DG813-893) parts of the
-dystroglycan
(DG) cytoplasmic domain were cotransfected, with rapsyn
domains fused with hSos or with hSos alone and grown for 4 days at
37 °C on plates containing galactose or glucose. No growth was seen
for the plates containing glucose (not shown).
-Dystroglycan Interacting with the RING-H2
Domain--
To further define the region within the
-dystroglycan
cytoplasmic domain that was involved in binding the rapsyn RING-H2 domain, we tested for interactions between rapsyn domains fused with
Sos and Myr-
-dystroglycan778-814, which encodes the membrane proximal domain, or Myr-
-dystroglycan813-893, encoding the membrane distal domain (Fig. 2B).
Myr-dystroglycan778-814 interacted with the rapsyn RING-H2
domain (Sos-rapsyn361-412) as well as with full-length
rapsyn (Sos-rapsyn5-412) but not with
rapsyn1-360-Sos, which lacks the RING-H2 domain. In
contrast, Myr-dystroglycan813-893 did not interact with full-length rapsyn or with rapsyn1-360-Sos, but when
expressed with the Sos-RING-H2 domain there was evidence of limited
yeast growth at 37 °C.
-dystroglycan appeared diffusely distributed at the
cell surface and not associated with rapsyn clusters when visualized by
a monoclonal antibody (Fig. 3,
a and b) or polyclonal antibody (Fig. 3,
c and d) directed against the
-dystroglycan
COOH-terminal 15 or 20 amino acids. For both antibodies,
-dystroglycan was detected only in transfected cells, and antibody
binding was not seen in fixed, nonpermeabilized cells (data not shown).
Similarly, when
-dystroglycan was visualized either by mAb IIH6
(Fig. 3, e and f) or mAb 1B7 (Fig. 3,
g and h),
-dystroglycan was distributed
diffusely at the surface and was not associated with rapsyn clusters.
For these antibodies, staining was also seen in nonpermeabilized,
transfected cells (data not shown). For these four antibodies,
dystroglycan immunostaining associated with rapsyn clusters was seen in
less than 1% of cells expressing both proteins.
View larger version (87K):
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Fig. 3.
Rapsyn clustering of dystroglycan in 293T
cells. A panel of antibodies directed against - or
-dystroglycan was used to detect dystroglycan coexpressed with
rapsyn in 293T cells. The primary and secondary antibodies were
incubated with the cells after fixation and permeabilization, as
described under "Experimental Procedures." The distributions of
rapsyn (left) and dystroglycan (right) were
visualized by the binding of the anti-rapsyn and anti-dystroglycan
antibodies indicated below. Dystroglycan immunostaining appeared
diffusely distributed at the surface and not associated with rapsyn
clusters when visualized by antibodies against the COOH terminus of
-dystroglycan (a, rabbit anti-rapsyn1-16;
b, mAb NCL-b- dystroglycan (NCL-b-DG);
c, rapsyn mAb 22F10; d, P20 antibody) or by
monoclonals specific for
-dystroglycan (e and
g, rabbit anti-rapsyn1-16; f, mAb
IIH6; h, mAb 1B7). Anti-dystroglycan signal was colocalized
with rapsyn clusters when it was visualized by the sheep polyclonal
FP-B directed against
/
-dystroglycan (i, mAb 22F10;
j, FP-B). Bar = 10 µm.
- or
-dystroglycan antibodies, and for cells
transfected with rapsyn only but incubated after permeabilization with
FP-B antibody and fluorescent secondary antibody, no fluorescence above
background was seen (data not shown).
View larger version (51K):
[in a new window]
Fig. 4.
Rapsyn TPRs 1 and 2 with the RING-H2 domain
are sufficient for dystroglycan clustering. a,
schematic representation of the rapsyn constructs. Borders of TPRs and
the RING-H2 domains are shown. The dashed lines represent
the deletions. b-g, 293T cells were transfected with
cDNAs encoding rapsyn mutants, along with dystroglycan. After
fixation and permeabilization, cells were incubated with primary
antibodies directed against rapsyn (mAb 22F10) and /
-dystroglycan
(FP-B) and the secondary antibodies. The distributions of rapsyn
(left) and dystroglycan (right) were visualized
with fluorescein isothiocyanate and rhodamine optics, respectively.
b and c, rapsyn1-287+351-412, containing TPRs 1-7 and the
RING-H2 domain formed clusters (b) and recruited
-dystroglycan to the clusters (c). d and
e, rapsyn1-254+351-412, containing TPRs 1-6,
clustered dystroglycan (e). f and g,
rapsyn1-90+351-412, containing TPRs 1-2 and the RING-H2
domain, formed clusters (f) that contained
-dystroglycan
(g) in >95% of cells expressing both proteins.
h-m, to test the ability of these rapsyn constructs to
cluster nAChRs, 293T cells were transfected with cDNAs encoding
rapsyn mutants, along with nAChR subunits. Fixed cells were incubated
with
-bungarotoxin, and then after permeabilization, cells were
incubated with mAb 22F10 and rabbit anti-
-bungarotoxin and then
secondary antibodies. The distributions of rapsyn (left) and
nAChRs (right) were visualized with fluorescein
isothiocyanate and rhodamine optics, respectively. nAChRs were
distributed uniformly at the cell surface in the presence of
rapsyn1-287+351-412 (h and i),
rapsyn1-254+351-412 (j and k), or
rapsyn1-90+351-411 (l and m).
Bar = 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan, for the rapsyn
RING-H2 domain. Previous studies based upon the properties of rapsyn
domains expressed in 293T cells (16, 18) established that the rapsyn
TPR domain is involved in self-association and that neither the
coiled-coil domain nor the RING-H2 domain, when expressed alone, was
able to associate with full-length, wild-type rapsyn. Furthermore, the
studies showed that the presence of the coiled-coil domain was required
for nAChR clustering. In the 293T expression system, however, nAChR
interactions with rapsyn could be identified only in the context of
rapsyn clustering, and it was not possible to determine whether the
rapsyn coiled-coil domain interacted with an nAChR domain in the
absence of the TPR domain.
-subunit,
or
-dystroglycan. Our results establish that the rapsyn domains can
each interact in an independent and selective manner with other protein
domains. The TPR domain is involved in self-association, and it did not
interact with other rapsyn domains or with the cytoplasmic domain of
the nAChR
-subunit or of
-dystroglycan. The rapsyn coiled-coil
domain, even in the absence of the rapsyn TPR domain, can interact with
the nAChR
-subunit cytoplasmic domain but not with the rapsyn
RING-H2 or TPR domain or with the
-dystroglycan cytoplasmic domain.
Rapsyn and the rapsyn coiled-coil domain can also interact with the
cytoplasmic domains of other nAChR subunits, but studies of these
interactions will be described in a later report.
-dystroglycan and that this domain cannot interact
with any of the domains within full-length rapsyn or with the nAChR
-subunit cytoplasmic domain. The interaction between the RING-H2
domain and the
-dystroglycan cytoplasmic domain can occur in the
absence of the rapsyn TPR or coiled-coil domain, and the RING-H2 domain
can function as an independent binding domain. Furthermore, based upon
the 293T assay, a rapsyn construct containing the minimal domain
structure necessary for self-association at the plasma membrane
(Myr-TPRs1+2) and the RING-H2 domain causes dystroglycan to be
clustered with rapsyn.
-dystroglycan or by a monoclonal or a polyclonal antibody specific
for the COOH terminus of
-dystroglycan. However, the clustering of
dystroglycan by rapsyn, as visualized by the FP-B antibody, was
unambiguous and seen in essentially all cells that expressed both
proteins. Based upon immunoblots, the affinity-purified FP-B antiserum
recognizes determinants in
- and
-dystroglycan (21). Since we do
not detect dystroglycan clustering with either antibody directed
against
-dystroglycan, we suspect that the majority of
-dystroglycan does not remain associated with
-dystroglycan on
the 293T cell surface. When
-dystroglycan was visualized with antibodies directed against the COOH-terminal 20 amino acids, there was
also no evidence of
-dystroglycan clustering by rapsyn. One possible
explanation for this is that the antibody (primary or secondary) cannot
bind when
-dystroglycan is in association with rapsyn. The fact that
rapsyn1-412 but not rapsyn1-412-GFP can
cluster dystroglycan demonstrates that modifications at the COOH
terminus of rapsyn also can interfere with dystroglycan clustering. It
also may be relevant that Tyr-890, the fourth amino acid before the
COOH terminus of dystroglycan, is a site of tyrosine phosphorylation that regulates dystrophin binding to
-dystroglycan (28). It remains
to be determined whether
-dystroglycan clustered with rapsyn is
phosphorylated at Tyr-890 and whether the antipeptide antibodies
directed against the
-dystroglycan COOH terminus recognize the
epitope when phosphorylated.
-Dystroglycan--
Sos-rapsyn interacted with
Myr-dystroglycan778-814 but not with
Myr-dystroglycan813-893. For the RING-H2 domain in
isolation (Sos-rapsyn361-412) the growth of yeast under selective conditions was also seen in the presence of
Myr-dystroglycan813-893, although growth was considerably
slower than in the presence of the
Myr-dystroglycan778-814. Further studies are required to determine whether these differences in growth reflect differences in
the affinity of interaction of the RING-H2 domain for determinants in
the two
-dystroglycan subdomains. Since Sos-rapsyn1-412 interacted only with the membrane proximal subdomain, it is that interaction that appears more biologically relevant.
-dystroglycan subdomain (dystroglycan778-813)
that binds to rapsyn does contain a proline triplet (Pro-807-809), it
is the membrane distal cytoplasmic domain
(dystroglycan813-893) that does not interact with rapsyn
that actually contains 22 prolines including prolines 887-889, which
are directly involved in binding to the dystrophin WW domain (30, 31)
and to the adaptor protein Grb2 (32). Further studies are required to
determine the surface of the rapsyn RING-H2 domain involved in
dystroglycan binding and to identify the determinants within
dystroglycan778-813 important for rapsyn binding.
-dystroglycan cytoplasmic domain in non-muscle cells, and there is
every reason to expect that this interaction occurs in muscle and is
important for the organization of nicotinic postsynaptic membrane. At
the adult neuromuscular junction dystroglycan is enriched in the
postsynaptic membrane, interacting with utrophin that is colocalized
with nAChRs and rapsyn in the primary gutter and with dystrophin in the
secondary folds (33). In addition, in experiments with cultured C2
myotubes, utrophin was found associated primarily with the large, but
not the small, nAChRs clusters, which suggested that interactions
between rapsyn/nAChR and proteins of the dystrophin
complex may be involved in the growth of nAChR clusters (34). Recently,
it has been shown (35) that for myotubes differentiated from
dystroglycan
/
embryonic stem cells and for neuromuscular junctions
of dystroglycan-deficient chimeric mice, the organization of nAChR
clusters is more fragmented than in wild-type muscle. In addition, in
the absence
-dystrobrevin, a dystrophin homolog that also binds to
-dystroglycan and is enriched with nAChRs in the post-synaptic
membrane, there is a similar failure of neuromuscular junction
maturation (36). These results emphasize that
/
-dystroglycan and
the link it forms between the extracellular matrix and the cytoskeleton
are not required for the initial formation of nAChR clusters but are
important for the maturation of the nAChR clusters into large, compact arrays.
-dystroglycan or other
dystrophin/utrophin-associated proteins. When coexpressed with nAChRs
in Xenopus oocytes, a mutant rapsyn containing substitutions within the RING-H2 domain that prevent zinc coordination was capable of
clustering nAChRs, but the clusters were smaller in size than for
wild-type rapsyn (37). Furthermore, injection of a fusion protein
containing the rapsyn RING-H2 domain into rat myotubes caused a
fragmentation of nAChR clusters (38) without any evidence that the
RING-H2 domain itself was interacting with rapsyn or nAChRs. Although
it remains to be determined whether the rapsyn RING-H2 domain binds to
proteins in addition to
-dystroglycan, these results in conjunction
with our data indicate that the interaction between the rapsyn RING-H2
domain and
-dystroglycan is crucial for the maturation of the nAChR
clusters. However, the size of the rapsyn clusters in 293T does not
increase in the presence of transfected dystroglycan, and there may be
other, as yet unidentified, proteins that interact with rapsyn and are
important for nAChR clustering. We are currently using the hSos
recruitment assay to look for interactions between rapsyn and other
proteins such as MuSK, the receptor tyrosine kinase that binds agrin
(39), that are known to be localized with nAChRs in the nicotinic
post-synaptic membrane and to screen libraries from skeletal muscle to
identify other proteins interacting with rapsyn.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Aronheim for the p5'Sos plasmid and for many useful protocols for the Sos recruitment assay. We also thank Drs. K. P. Campbell and S. Carbonetto for the dystroglycan antibodies and plasmids and Dr. T. C. Petrucci for dystroglycan antibody P-20. We thank J. Trinidad and M. Bianchetta for useful comments about the manuscript.
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FOOTNOTES |
---|
* This research was supported in part by United States Public Health Service Grant NS 18458.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed:, Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen@hms.harvard.edu.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M103258200
2 M. Bartoli, unpublished information.
3 J. Cohen, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: nAChR, nicotinic acetylcholine receptor; TPR, tetratricopeptide repeat; hSOS, human Son of Sevenless; PCR, polymerase chain reaction; mAb, monoclonal antibody; GFP, green fluorescent protein.
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