From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 30, 2000
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ABSTRACT |
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Rapsyn, a 43-kDa peripheral membrane protein of
skeletal muscle, is essential for clustering nicotinic acetylcholine
receptors (nAChR) in the postsynaptic membrane. Previous studies with
rapsyn NH2-terminal fragments fused to green
fluorescent protein, expressed in 293T cells along with nAChRs,
establish the following: Rapsyn-(1-90), containing the
myristoylated amino terminus and two tetratricopeptide repeats (TPRs),
was sufficient for self-association at the plasma membrane;
rapsyn-(1-287), containing seven TPRs, did not cluster nAChRs; whereas
rapsyn-(1-360), containing a coiled-coil domain (rapsyn-(298-331)), clustered nAChRs. To further analyze the role of
rapsyn structural domains in self-association and nAChR clustering, we
have characterized the clustering properties of additional rapsyn
mutants containing deletions and substitutions within the TPR and
coiled-coil domains. A mutant lacking the coiled-coil domain alone
(rapsyn-( A characteristic and important feature of the vertebrate
neuromuscular junction is the clustering of nicotinic acetylcholine receptors (nAChR)1 at high
surface density (~10,000/µ2) in the post-synaptic
membrane underlying the nerve terminal, with the nAChR surface density
decreasing by a factor of 100-1000 within a few microns. Rapsyn, a
peripheral membrane protein of muscle, plays a critical role in the
clustering of nAChRs and in the organization of the
postsynaptic cytoskeletal complex (for a recent review, see Ref. 1).
Mutant mice lacking rapsyn show severe neuromuscular abnormality marked
by the absence of nAChR clusters in the postsynaptic membrane as well
as the absence of cytoskeletal components such as utrophin and
dystroglycan (2).
When expressed in nonmuscle cells, rapsyn forms membrane-associated
clusters and recruits nAChRs to these clusters (3-6). Furthermore,
when coexpressed in fibroblasts rapsyn also clusters The three-dimensional structure of rapsyn is not known. However, rapsyn
primary structure suggests the presence of distinct structural domains
(summarized in Fig. 1). The amino
terminus of rapsyn is myristoylated (12, 13). Rapsyn-(6-319) is
predicted to form 8 tetratricopeptide repeats (TPRs) (14), while
rapsyn-(298-331) contains a putative coiled-coil domain (6). The
COOH-terminal cysteine-rich domain of rapsyn between amino acids 363 and 402 is predicted to be a RING-H2 domain (15). Rapsyn amino acids 403-406 contain a consensus sequence for phosphorylation by both protein kinase A and protein kinase C.
288-348)), failed to cluster nAChRs. Within the
coiled-coil domain neutralization of the charged side chains was
tolerated, while alanine substitutions of large hydrophobic residues
resulted in the loss of nAChR clustering. Rapsyn self-association requires at least two TPRs, as a single TPR (TPR1 or TPR2 alone) was
not sufficient. While TPRs 1 and 2 are sufficient for self-association, they are not necessary, as TPRs 3-7 also formed clusters similar to
wild-type rapsyn. Fragments containing TPRs co-localized with full-length rapsyn, while the expressed coiled-coil or RING-H2 domain
did not. These results are discussed in terms of a homology model of
rapsyn, based on the three-dimensional structure of the TPR domain of
protein phosphatase 5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan,
the integral membrane protein of the dystrophin complex (7), as well as
the agrin receptor, the receptor tyrosine kinase MuSK (8-10).
Biochemical studies provide evidence for a direct interaction between
rapsyn and the cytoplasmic domain of
-dystroglycan (11).
View larger version (9K):
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Fig. 1.
Schematic diagram of the structural domains
of rapsyn. Indicated are the myristoylated NH2
terminus (N-myr) and the borders of the seven putative TPRs,
the coiled-coil domain, the RING-H2 domain, and the consensus sequence
for phosphorylation.
We recently provided a first characterization of the rapsyn structural domains involved in membrane targeting, self-association, and nAChR clustering (6). Our results indicated that rapsyn NH2-terminal fatty acylation is both required and sufficient for membrane targeting and that rapsyn-(1-90), containing two TPR domains, is sufficient for self-association. In addition, while rapsyn-(1-360) clustered nAChRs, rapsyn-(1-287) did not, indicating that rapsyn-(287-360) is important for nAChR clustering. Within rapsyn-(287-360) we identified a putative coiled-coil domain, rapsyn-(298-331), and showed that disruption of the coiled-coil propensity by alanine insertions prevented nAChR clustering. These results indicated that the COOH-terminal rapsyn RING-H2 domain and the adjacent phosphorylation sites were not essential for either rapsyn or nAChR clustering.
We report here further mutational analyses of rapsyn to elucidate the
role of the coiled-coil domain in nAChR clustering and the role of the
TPRs in rapsyn self-association. These rapsyn mutants, fused at their
COOH termini to GFP, were expressed transiently in 293T cells along
with nAChR subunits and were visualized by fluorescence microscopy
24-36 h after transfection. The results indicate that nAChR clustering
depends critically on the structure of the hydrophobic surface of the
coiled-coil domain but not on the hydrophilic surface. While TPRs
mediate rapsyn self-association, nAChR clustering is retained in
mutants lacking as many as four TPRs.
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EXPERIMENTAL PROCEDURES |
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Materials and Methods
All restriction enzymes were purchased from New England Biolabs except Bpu1102I (Life Technologies, Inc.). 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 template pGL-rapsyn (mouse rapsyn subcloned into pGreenLantern vector; Life Technologies, Inc.), 50-100 pmol of each primer, 250 µM each dNTP, and 5 units of Pfu DNA polymerase (Stratagene) for 30 cycles at 94 °C for 2 min, 50 °C for 1 min, and 72 °C for 1 min. All constructs were tested both by restriction enzyme analysis and by sequencing across the full-length of the inserted fragments.
Plasmid Construction and Mutagenesis
Rapsyn-(1-41)-GFP-- This construct encodes rapsyn amino acid sequence 1-41 containing the consensus sequence for N-myristoylation and TPR1. Primers GACACTATAGAAGGTACG and GCAAGTACTGCCCACGAGGTCAGAGCCCTT were used along with the template pGL-rapsyn-(1-412)-GFP (6) in a PCR to amplify rapsyn amino acids 1-41. The PCR product with SpeI and ScaI ends was subcloned into pBK-CMV vector (Stratagene). The resulting plasmid was digested with SpeI and NotI, and the fragment containing the rapsyn sequence was subcloned into pGL vector lacking the GFP insert (made by digesting pGreenLantern with NotI, removing the GFP insert, and religating the vector fragment). GFP cDNA was then inserted at the NotI site to result in pGL-rapsyn-(1-41)-GFP.
Rapsyn-(1-15,33-90)-GFP-- This construct encodes rapsyn amino acid sequence 1-15, which contains the consensus sequence for N-myristoylation fused in-frame to amino acids 33-90 (TPR2). Primers GTGTGGATGAAGCAGCTGGAGAAGGGCTCT and GCCATCCAGTTCCACGAG were used along with pGL-rapsyn-(1-90)-GFP (6) as a template in a PCR to amplify rapsyn amino acids 33-90. The underlined letters denote the PvuII site in rapsyn. The resulting PCR product was digested with PvuII and NotI, followed by ligation with pGL-rapsyn-(1-15), which was obtained by digesting pGL-rapsyn with PvuII and NotI. GFP cDNA was then inserted at the NotI site, resulting in pGL-rapsyn-(1-15,33-90)-GFP.
Rapsyn-(16-90)-GFP--
This construct encodes full-length
rapsyn with a deletion of amino acids 16-90 (TPRs 1 and 2). The
cDNA fragment of mouse rapsyn from PvuII
(43) to BstUI (269) was deleted. Following digestion of
pGL-rapsyn-(1-412)-GFP with PvuII, the vector and insert
fragments were isolated. The insert fragment consisting of rapsyn
sequence was further digested with BstUI and
NotI. The vector fragment was digested with NotI.
The vector fragment with PvuII-NotI ends was then
ligated with the insert fragment with BstUI-NotI ends, resulting in pGL-rapsyn-(
16-90). GFP cDNA was then inserted at the NotI site to generate pGL-rapsyn-(
16-90)-GFP.
Rapsyn-(91-254)-GFP--
This construct encodes full-length
rapsyn with a deletion of amino acids 91-254 (containing TPRs 3-6).
The cDNA fragment of mouse rapsyn from BstUI
(269) to EcoRV (760) is deleted. Following digestion of
pGL-rapsyn-(1-412)-GFP with SpeI and EcoRV, the
vector and insert fragments were isolated. The insert fragment
consisting of rapsyn sequence was further digested with
BstUI. The vector fragment with SpeI and
EcoRV ends was then ligated with the insert fragment with
SpeI-BstUI ends, resulting in
pGL- rapsyn-(
91-254)-GFP.
Rapsyn-(194-254)-GFP--
This construct encodes full-length
rapsyn with a deletion of amino acids 194-254 (TPR 6). The cDNA
fragment of mouse rapsyn from HincII (577) to
EcoRV (760) is deleted. Following digestion of
pGL-rapsyn-(1-412)-GFP with SpeI and EcoRV, the
vector and insert fragments were isolated. The insert fragment
consisting of rapsyn sequence was further digested with
HincII and ligated with the vector fragment with
SpeI-EcoRV ends, resulting in
pGL-rapsyn- (
194-254)-GFP.
Rapsyn-(255-287)-GFP--
This construct encodes full-length
rapsyn with a deletion of amino acids 255-287 (TPR 7). The cDNA
fragment of mouse rapsyn from EcoRV (760) to
PmlI (859) is deleted. pGL-rapsyn-(1-412)-GFP was digested
with EcoRV and PmlI. The vector fragment
consisting of rapsyn sequence was isolated and religated, resulting in
pGL-rapsyn-(
255-287)-GFP.
Rapsyn-(288-348)-GFP--
This construct encodes full-length
rapsyn with a deletion of amino acids 288-348 which contain the
coiled-coil domain (6). The cDNA fragment of mouse rapsyn between
BsaAI sites (859 and 1042) is deleted. Mouse rapsyn cDNA
was subcloned into pSVL vector (Amersham Pharmacia Biotech).
pSVL-rapsyn was then digested with BsaAI and the vector
fragment containing rapsyn sequence was isolated and religated,
resulting in pSVL-rapsyn-(
288-348). pBK-CMV (Stratagene) was used
as a shuttle vector to subclone rapsyn-(
288-348) into pGL vector,
and GFP cDNA was then inserted at the NotI site,
resulting in pGL-rapsyn-(
288-348)-GFP.
Rapsyn-(1-340)-GFP-- This construct, which encodes rapsyn amino acids 1-340, terminates immediately after the coiled-coil domain and lacks the RING-H2 domain as well as the 20 amino acids between the coiled-coil and the RING-H2 domains. This construct differs from rapsyn-(1-360)-GFP (6), which includes these 20 amino acids. Primers GATGGGCCCGCGGCCGCTCTAGAAGTCCCTTTGCTGCGGTAGATGCTCTCACT and TACGACTCCGCTATGAGC were used along with pGL-rapsyn as a template in a PCR to amplify rapsyn cDNA sequence from 805-1020. The underlined letters denote the introduced XbaI site. The resulting PCR product was digested with PmlI and XbaI, and it was substituted in place of the PmlI-XbaI fragment of pGL-rapsyn-GFP, resulting in pGL-rapsyn-(1-340)-GFP.
Rapsyn-(1-15,91-287)-GFP--
This construct encodes a rapsyn
mutant that lacks TPRs 1 and 2, the coiled-coil domain, and the RING-H2
domain. The NH2-terminal sequence of
pGL-rapsyn-(1-287)-GFP (6) between SpeI and
EcoRV was removed and replaced with that from
pGL-rapsyn-(16-90)-GFP.
Rapsyn-(1-15,91-340)-GFP--
This construct encodes a rapsyn
mutant that lacks TPRs 1 and 2 and the RING-H2 domain, but includes the
coiled-coil domain. The NH2-terminal sequence of
pGL-rapsyn-(1-340)-GFP between SpeI and EcoRV
was removed and replaced with that from pGL-rapsyn-(16-90)-GFP.
Rapsyn-(287-340)-GFP-- This construct encodes the coiled-coil domain of rapsyn in fusion with GFP. Primers AGATAAAATAGAATAACTAGTGCCACCATGCACGTGCTGCTGGGTGTGGCCAAGTGCTGGATG and GCCATCCAGTTCCACGAG were used along with pGL-rapsyn-(1-340)-GFP as a template in a PCR to amplify rapsyn cDNA sequence from 859 to 1020. The resulting PCR product was digested with SpeI and XbaI and then inserted into pGL vector.
Rapsyn-(G2A-360)-GFP-- This construct encodes for rapsyn amino acids G2A-360, lacking the consensus sequence for N-myristoylation and the cysteine-rich RING-H2 domain. pGL-rapsyn-(G2A)-GFP and pGL-rapsyn-(1-360)-GFP (described previously (6)) were digested with SpeI and EcoRV. The NH2-terminal sequence (SpeI-EcoRV) of rapsyn was swapped with that of rapsynG2A resulting in pGL-rapsyn-(G2A-360)-GFP.
Rapsyn-(351-411)-GFP-- This construct encodes the RING-H2 domain of rapsyn fused with GFP. Primers AGATAAAATAGAATAACTAGTGCCACCATGGTAGTGAGGTTCCACGAGTGCGTGGAGGAGACTGAG and GCCATCCAGTTCCACGAG were used along with pGL-rapsyn-(1-412)-GFP as a template in a PCR to amplify rapsyn cDNA sequence from 1045 to 1236. The underlined letters denote the introduced SpeI site. The resulting PCR product was digested with SpeI and XbaI, and then inserted into pGL vector resulting in pGL-rapsyn-(351-411)-GFP.
Rapsyn-(298-331)-LIV A-GFP--
This construct encodes a
full-length rapsyn mutant with Ala replacements of the hydrophobic
residues (Leu, Ile, and Val) within rapsyn-(298-331), the coiled-coil
domain. Mutations were introduced by sequential PCR steps. The
first PCR was done using the primers AGCCCTGTTCTTTCCCTG and
GTCCTGGGCTTTCTCAGCGGCATCCGCAGCCTTGTCTTGCGCCTTCCGGGCCATCCA with pGL-rapsyn-GFP as a template. The highlighted codons represent Ala
replacements of Ile309 (12), Leu306 (9), and
Val301 (4) where the numbers in parentheses refer to the
position within the coiled-coil domain with number 1 being rapsyn-(298) (Ala). The second PCR was done using the primers
GAGAAAGCCCAGGACGCAGCTGAAGAGGCTGGCAATAAGGCGAGCCAGGCCAAGGCGCATTGCGCGAGTGAGAGCATCTA and ACCACGCCAGTGAACAGT with pGL-rapsyn-GFP as a template. In the above primers, codons written in small letters represent the
overlapping sequence between the two PCR products. The highlighted
codons represent Ala replacements of Leu315(18),
Val319 (22), Leu323 (26), Leu326
(29), Leu328 (31), and Leu331 (34), with the
number in the parentheses corresponding to the numbers used in Fig.
3a. The two fragments encompassing the mutation were
annealed with each other, extended by mutually primed synthesis, and
amplified by a second PCR. The resulting fragment was then digested
with PmlI and XbaI and then substituted in place
of the corresponding wild-type rapsyn sequence in pGL-rapsyn-GFP.
Rapsyn-(298-331)-ED QN-GFP--
This construct encodes a
full-length rapsyn with the Glu and Asp within rapsyn-(298-331)
replaced by Gln and Asn, respectively. Primers
GGGCAGGTGCACGTGCTGCTGGGTGTGGCCAAGTGCTGGATGGCCCGGAAGGTGCAAAACAAGGC, with the D303N (6) mutation, and
CAGCTTGAGCTGGCTCAGCTTATTGCCAAGCTGTTGAGCTAAGTTCTGGGCTTTCTGAATGGCATTCAAAGC, with E318Q (21), E317Q (20), D314N (17), E310Q (13), and D307N
(10) mutations, were employed in a PCR along with the template
pGL-rapsyn. The resulting PCR product was digested with PmlI
and Bpu1102I and then substituted in place of the
corresponding wild-type rapsyn sequence in pGL-rapsyn-GFP.
Rapsyn-(298-322)-KR Q-GFP--
This construct encodes a
full-length rapsyn with the lysine and arginine residues within
rapsyn-(298-322) replaced by Gln. Primers
GGGCAGGTGCACGTGCTGCTGGGTGTGGCCAAGTGCTGGATGGCCCAGCAGGTGCAAGACCAGGCTTTGGAT, with R299Q (2), K300Q (3), K304Q (7) mutations, and
CAGCTTGAGCTGGCTCAGCTGATTGCCAACCTCTTCAGCTAAGTCCTGGGCTTGCTCAATGGC, with K322Q (25) and K311Q (14) mutations were employed in a PCR
with pGL-rapsyn as template. The resulting PCR product was digested
with PmlI and Bpu1102I and then substituted in
place of the corresponding wild-type rapsyn sequence in
pGL-rapsyn-GFP.
Expression of Rapsyn, Rapsyn Mutants, and nAChR in 293T Cells
Transfection of 293T cells by the calcium phosphate method, cell
staining, and immunofluorescence experiments were done as described
(6), except that the staining was done on cells fixed for 20 min with
2% paraformaldehyde followed by an incubation with a buffer containing
PBS, 10% calf serum, 4% bovine serum albumin, and 100 mM L-lysine for 1 h. Under these
conditions, -bungarotoxin binding to surface nAChRs was similar to
that of unfixed cells, and the distribution of nAChRs on the cell
surface was generally uniform with submicron granularity. Most of the rapsyn clusters ranged from 1 to 5 µ in size with occasional larger plaques of 5-10 µ. Cells were visualized using a Nikon Eclipse E800
epifluorescence microscope with a Nikon 100X Plan Fluor objective (NA1.3). Green and red fluorescence were visualized through Nikon 91617 (excitation, 480 ± 20 nm; emission, 515-560 nm) and 96171 filters (excitation, 540 ± 10 nm; emission, 590-650 nm),
respectively. In some experiments photographs were taken with Kodak
160T film and digitized. Most of the images were acquired with a
Micromax CH250 CCD camera (Princeton Instruments) using MetaMorph
software. Figures were prepared from the digitized images using Adobe Photoshop.
Anti-rapsyn monoclonal MAb19F4A, used in the identification of rapsyn in Fig. 6, was prepared by using Torpedo rapsyn as immunogen (16). Based upon epitope mapping studies it has been shown to recognize rapsyn amino acid sequence 396-411.2 In our immunofluorescence studies, it did not recognize rapsyn-(1-360) or other smaller fragments (data not shown).
Quantification of Cells with Rapsyn or nAChR Clusters
In general, within each cell expressing both rapsyn and nAChR,
not all rapsyn clusters are associated with nAChRs. This is expected
since only the surface nAChRs are labeled with -bungarotoxin, while
rapsyn-GFP is distributed at the plasma membrane and also intracellularly. For experiments involving expression of rapsyn-GFP (or
mutants) and nAChRs in 293T cells, quantification of nAChR clusters
relative to rapsyn clusters was done as follows. In each experiment,
100 cells positive for both rapsyn and nAChR expression were
identified. For these we quantified the number of cells with nAChRs
that co-localized with rapsyn clusters and the number of cells with
nAChRs distributed diffusely. In experiments involving rapsyn mutants
with TPR deletions, in some cells nAChRs were seen both clustered with
rapsyn and also distributed diffusely on the surface. For
quantification, these cells were classified as containing clustered
nAChRs. Each experiment was repeated at least four times, cells were
scored as above, and the results are presented as the % of cells with
nAChR clusters (mean and S.D.).
Molecular Modeling of Rapsyn
All molecular modeling studies were done with Insight II,
Version 98 (MSI, San Diego, CA) on a Silicon Graphics IRIS work station. The mouse rapsyn TPR domain was homology modeled based upon
the three-dimensional crystal structure of the TPR domain of protein
phosphatase 5 (17). The coordinates of this domain, containing 3 TPRs,
were obtained from the Brookhaven Protein Data Base (PDB number 1A17).
The sequences of the rapsyn TPRs 1-7 (14) were used sequentially to
create the model. TPRs 1-3 and 4-6 were each modeled individually,
and then they were combined in register. TPR 7, initially modeled with
TPR 8, was then added. For rapsyn, the intervening sequences between
TPRs 2 and 7 each contain 6-9 amino acids, while in protein
phosphatase 5 there are no residues between TPRs. For rapsyn random
loops were generated for these sequences, and these loops were then
fused with the TPR structures. Rapsyn amino acids 286-331 were modeled
as an energy minimized, single long helix, based on our previous
prediction that rapsyn 298-331 has a high propensity to be organized
as an -helical coiled-coil (6). The orientation of the coiled-coil domain with respect to the TPRs was done arbitrarily and represents only one of the possibilities. The entire rapsyn model was then energy
minimized using the Discover module, first with steepest descent
algorithm and then by conjugate gradient algorithm until the energy of
the entire molecule was low.
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RESULTS |
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Rapsyn Coiled-coil Domain Is Essential for Clustering
nAChRs--
Previously we had shown (6) that rapsyn-(1-287)-GFP could
self-associate but not cluster nAChRs, while rapsyn-(1-360)-GFP could
cluster nAChRs. Furthermore, rapsyn amino acids 298-331 were predicted
to have a high propensity to form an -helical coiled-coil, and
alanine insertions within this region that disrupted coiled-coil
propensity also disrupted rapsyn's ability to cluster nAChRs without
affecting rapsyn self-association. To further assess the importance of
the rapsyn coiled-coil domain in nAChR clustering, we made two
additional constructs: (i) rapsyn-(1-340)-GFP, with the rapsyn
sequence terminated immediately after the coiled-coil domain; and (ii)
rapsyn-(
288-348)-GFP, full-length rapsyn with the coiled-coil
domain deleted (Fig. 2a). When
expressed in 293T cells, rapsyn-(1-340)-GFP formed distinct
membrane-associated clusters in all transfected cells and clustered
nAChRs at the cell surface (Fig. 2, b and c) in
95 ± 5% of cells expressing both proteins. The appearance of
rapsyn/nAChR clusters was indistinguishable from that of wild-type
rapsyn. In contrast, the construct lacking the coiled-coil domain,
rapsyn-(
288-348)-GFP, formed membrane-associated clusters similar
to wild type but did not cluster nAChRs (Fig. 2, d and
e). When nAChR distribution was quantified in six separate transfection experiments, nAChRs were clustered with rapsyn in only
8 ± 2% of cells. These results establish that the coiled-coil domain is essential for nAChR clustering, while the adjacent amino acids 340-360 of rapsyn are not necessary.
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The Structure of Hydrophobic Face of the Rapsyn Coiled-coil Domain
Is Important for nAChR Clustering--
Rapsyn-(298-331) has a high
propensity to organize as a coiled-coil domain, since it can form a
10-turn amphipathic -helix with a hydrophobic moment of 0.6 (6)
(Fig. 3a). When modeled as a
right-handed
-helix, the amphipathic character of the helix is
clearly visualized with a hydrophobic face (Fig. 3b, left) and a hydrophilic face (Fig. 3b, right). Striking features
of the hydrophilic surface are the presence of a strip of six-acidic side chains forming a continuous surface over four helix turns and the
presence of 5 lysine residues spaced to form a lysine ladder. These
lysines are conserved in rapsyn from different species. The hydrophobic
surface, which extends over 10 turns, consists of 5 alanine residues in
the first four helical turns and nine bulky side chains (Leu, Ile, and
Val).
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To analyze the importance of the particular amino acids that contribute
to the hydrophobic and hydrophilic helix surfaces, three constructs
with point mutations were tested (Fig.
4a): (i) rapsyn-(298-331)-ED
QN-GFP, full-length rapsyn fused to GFP with the 3 Glu and 2 Asp
within amino acids 298-331 mutated to Gln and Asn, respectively; (ii)
rapsyn-(298-322)-KR
Q-GFP, with the 4 Lys and 1 Arg within amino
acids 298-322 all replaced by Gln; (iii) rapsyn-(298-331)-LIV
A-GFP, with the 6 Leu, 1 Ile, and 2 Val within amino acids 298-331 all
replaced by alanine. None of these substitutions altered the
coiled-coil propensity as calculated by the COILS program (18) (data
not shown). When expressed in 293T cells, rapsyn-(298-331)-ED
QN-GFP formed distinct clusters in all transfected cells. nAChRs were
clustered with rapsyn at the surface (Fig. 4, b-d) in
72 ± 7% cells expressing both proteins and were diffusely
distributed in the other cells. Similarly, rapsyn-(298-322)-KR
Q-GFP also formed clusters in all transfected cells, and nAChRs were
associated with these clusters (Fig. 4, e-g) in 81 ± 11% cells expressing both proteins. In contrast, while
rapsyn-(298-331)-LIV
A-GFP did form clusters at the cell surface
in all the cells, only in 17 ± 8% of these cells were nAChRs
associated with any of these clusters. In a representative cell (Fig.
4, h-j) nAChRs are not associated with rapsyn clusters although there is a microgranular distribution of nAChRs in some parts
of the cell surface (see also Ref. 6). These results establish that
despite the conservation of the charged residues in rapsyn-(298-331),
their presence is not necessary for nAChR clustering. In contrast,
alteration of the structure of the hydrophobic surface results in the
lack of nAChR clustering.
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Rapsyn Self-association Requires Two TPRs--
Previous results
had shown that rapsyn-(1-90) containing the myristoylated amino
terminus and the first two TPRs was sufficient for rapsyn
self-association (6). To identify the minimal structural requirements
for rapsyn self-association, we also created chimeric proteins
consisting of rapsyn-(1-41), encoding N-Myr and TPR1, or
rapsyn-(1-15,33-90) encoding N-Myr and TPR2, each fused at its COOH
terminus to GFP. Furthermore, to analyze the specific requirements of
TPRs 1 and 2 in rapsyn self-association, we created rapsyn-(16-90)-GFP, which encodes a full-length rapsyn with TPRs 1 and 2 deleted, as well as rapsyn-(1-15, 91-287)-GFP, encoding TPRs
3-7, and rapsyn-(1-15, 91-340)-GFP, containing TPRs 3-7 and the
coiled-coil domain (Fig. 5a).
When expressed in 293T cells, rapsyn-(1-90)-GFP formed distinct
clusters (Fig. 5b). However, constructs containing
myristoylated TPR1 (rapsyn-(1-41)-GFP) or TPR2 (rapsyn-(1-15,
33-90)-GFP), although at least in part targeted to the plasma
membrane, failed to form any clusters in more than 98% of transfected
cells (Fig. 5, c and d, respectively). In all the
cells expressing rapsyn-(1-41)-GFP or rapsyn-(1-15, 33-90)-GFP, in
addition to fluorescence at the plasma membrane, a fraction was from
within the cell. Rapsyn-(
16-90)-GFP, which lacked TPRs 1 and 2 but
contained TPRs 3-7 and the coiled-coil and RING-H2 domains, formed
distinct clusters similar to wild-type rapsyn in all cells (Fig.
5e). Rapsyn self-association was clearly a property of the
TPR domain, since similar clustering was also seen for rapsyn-(1-15,
91-287)-GFP (Fig. 5f) and for rapsyn-(1-15, 91-340)-GFP
(Fig. 5g).
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The Interaction of Rapsyn Domains with Wild-type Rapsyn--
We
also characterized the capacity of constructs encoding rapsyn domains
to associate with wild-type, full-length rapsyn clustered at the cell
surface. The distribution of rapsyn domains fused to GFP was visualized
by fluorescein isothiocyanate optics, while wild-type rapsyn was
visualized by the binding of rhodamine-conjugated goat anti-mouse
antibody to mAb 19F4A, a mouse monoclonal that recognizes rapsyn of
many species (16). Based upon epitope mapping studies with
Torpedo rapsyn, mAb194A recognizes a COOH-terminal epitope
(rapsyn-(396-411)),3 and
based upon immunofluorescence it did not recognize rapsyn-(1-360) or
other smaller fragments expressed in 293T cells (data not shown). When
coexpressed with full-length rapsyn, rapsyn-(1-15)-GFP was distributed
diffusely at the cell surface, but it was not associated with the
rapsyn clusters (Fig. 6, a and b).
Rapsyn-(1-90)-GFP, containing TPRs 1 and 2, formed clusters, as
previously shown (Fig. 5b and Ref. 6), and these clusters
were co-localized with the clusters of full-length rapsyn (Fig. 6,
c and d). Rapsyn-(G2A-360)-GFP, which lacks the
myristoylation consensus sequence and the RING-H2 domain but contains
TPRs 1-7 and the coiled-coil domain, formed clusters co-localized with
the rapsyn clusters (Fig. 6, e and f).
Rapsyn-(287-340)-GFP, comprising the coiled-coil domain, was distributed diffusely in the cell and did not associate with rapsyn clusters (Fig. 6, g and h).
Rapsyn-(351-411)-GFP, containing the RING-H2 domain, was distributed
diffusely throughout the cell and was enriched in the nucleus, but it
did not associate with rapsyn clusters (Fig.
6, i and j) similar
to the epitope-tagged rapsyn RING-H2 domain (19). These results further
demonstrate that rapsyn self-association is specifically mediated by
the TPR domain. The GFP-tagged fragments of rapsyn containing only the coiled-coil domain, the RING-H2 domain, or the myristoylated amino terminus were not recruited to clusters of wild type rapsyn at the cell
surface, while rapsyn-(1-90) or the nonmyristoylated rapsyn-(G2A-360)
both associated with full-length rapsyn.
|
Rapsyn TPRs 1-7 and nAChR Clustering--
To further analyze the
role of the rapsyn TPR domain in nAChR clustering, a series of
constructs with internal deletions corresponding to specific TPRs were
created and expressed in 293T cells along with nAChRs (Fig.
7a). nAChRs were clustered by
rapsyn-(16-90)-GFP, the construct lacking the first two TPRs, in
62 ± 6% of cells expressing both proteins, with nAChRs
distributed uniformly in other cells. Fig. 7, b and
c, show representative images of a cell containing clustered
nAChRs, and Fig. 7, d and e, show a cell where
nAChRs, distributed on the surface with some granularity, are not
associated with the rapsyn clusters. Similarly,
rapsyn-(
91-254)-GFP, a construct lacking TPRs 3-6, formed distinct
clusters in all the cells, with nAChRs co-clustered (Fig. 7,
f and g) in 44 ± 18% of cells. Other
rapsyn mutants lacking either TPR6 (rapsyn-(
194-254)-GFP; Fig. 7,
h and i) or TPR7 (rapsyn-(
255-287)-GFP; Fig.
7, j and k) were clustered in all the cells but
clustered nAChRs in 52 ± 11 and 41 ± 15%, respectively.
These results indicate that a mutant rapsyn containing as few as three
TPRs (TPRs 1, 2, and 7) is able to cluster nAChRs but that neither TPR
1, 2, or 7 is required. However, for these TPR deletion mutants there
were two populations of cells, those with nAChR clusters and those
without. Further studies are required to identify the differences
between these two groups.
|
Nuclear Localization of Non-myristoylated
Rapsyn--
Rapsyn-(G2A)-GFP, with the amino-terminal glycine mutated
to alanine to prevent N-myristoylation, is preferentially
targeted to the nucleus (6, 20). To determine whether it is the
COOH-terminal region of rapsyn containing the RING-H2 domain that
contributes to the nuclear retention of rapsyn-(G2A)-GFP, we examined
the distribution in 293T cells of rapsyn-(G2A-360)-GFP that contained the G2A mutation and lacked the RING-H2 domain (amino acids 361-412; Fig. 8a). When coexpressed
with nAChRs in 293T cells, as expected, rapsyn-(G2A)-GFP was targeted
to the nucleus, while nAChRs were distributed diffusely on the plasma
membrane (Fig. 8, b and c). In contrast,
rapsyn-(G2A-360)-GFP was distributed in the cytoplasm and excluded from
the nucleus, with occasional enrichment at the surface. Most of the
nAChRs appeared uniformly distributed on the cell surface (Fig. 8,
d and e).
|
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DISCUSSION |
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The results presented here extend our understanding of the role of
the rapsyn coiled-coil and TPR domains in nAChR clustering and
self-association, respectively. Internal deletion of the rapsyn sequence corresponding to the coiled-coil domain (rapsyn-(288-348)) completely abolished nAChR clustering without affecting rapsyn self-association. This result further emphasizes that the rapsyn coiled-coil domain is the single most important domain required for
nAChR clustering. While the COOH terminus of rapsyn containing the
RING-H2 domain may be a determinant of the size of rapsyn and nAChR
clusters (21), no nAChR clustering was seen for a rapsyn mutant
containing seven TPRs and the RING-H2 domain but lacking the
coiled-coil domain.
The Rapsyn Coiled-coil Domain and nAChR Clustering-- Mutational analyses of the hydrophobic and hydrophilic surfaces of the rapsyn coiled-coil domain establish that it is the specific structure of the hydrophobic surface that is crucial for rapsyn to cluster nAChRs (Fig. 4). A classical coiled-coil domain is characterized by a heptad repeat sequence (a b c d e f g)n in which amino acids at positions a and d are typically hydrophobic, residues at e and g are often charged, and those at the solvent-exposed b, c, and f positions are predominantly polar. In the rapsyn coiled-coil domain there is a continuous hydrophobic surface (Fig. 3b) defined by side chains at position d (Ala1, Ala8, Ala15, Val22, and Leu29) and position a (Ile12, Ala19, and Leu26), along with Val4 and Leu18 (position g) and Leu9 (position e). The structure of this surface is essential for nAChR clustering, since mutations of the nine large hydrophobic residues in this region, some of which are conserved across species, to alanine resulted in the loss of nAChR clustering. While this result demonstrates the importance of the hydrophobic surface, additional point mutations are necessary to define the contribution made by individual amino acids in this region.
It was surprising that either the charge-neutralizing substitutions of the highly conserved positively charged residues (lysine ladder) or the negatively charged residues had no significant effect on nAChR clustering. While our results suggest clearly that charges are not required at these positions, it is possible that the size, hydrophilicity, or hydrogen bonds forming capabilities of the side chains at these positions are important. However, the high degree of charge conservation at these positions in rapsyn from species ranging from human to Caenorhabditis elegans suggests that these side chains may be involved in ionic interactions contributing to the stability of a helix bundle as seen in other homo-oligomeric or hetero-oligomeric coiled-coil structures (22, 23). Significantly, rapsyn-(287-340)-GFP, which contains only the coiled-coil domain, did not interact with full-length rapsyn. This result indicates that the coiled-coil domain is unlikely to self-associate, and it is consistent with the hypothesis that the rapsyn coiled-coil domain associates with a coiled-coil domain in the nAChR.
There is growing evidence for direct interaction between rapsyn and
nAChRs. Early chemical cross-linking studies and freeze fracture
immunoelectron microscopy both supported the view that rapsyn is in
close proximity to the cytoplasmic domains of nAChR (24, 25) (for a
review, see Ref. 26). The 4.6-Å structure recently determined by
electron microscopy of tubular crystals of Torpedo
postsynaptic membranes included on the cytoplasmic surface electron
dense regions immediately underneath the nAChR which were attributed to
rapsyn (27). nAChR subunits expressed individually in nonmuscle cells
can be clustered by rapsyn (28), and recent studies indicate that this
clustering is mediated by the large cytoplasmic loop between the M3 and
M4 transmembrane regions of the nAChR subunit (29). In addition,
overlay experiments with 125I-rapsyn suggest that rapsyn
binds to itself with high affinity and to full-length nAChR
,
,
or
subunit, but not to truncated
or
subunit lacking the
cytoplasmic loop (30).
Multiple Determinants in the TPR Domain Mediate Rapsyn Self-association-- Analysis of the clustering properties of rapsyn mutants containing deletions within the predicted TPR domain as well as the clustering properties of other GFP-tagged rapsyn domains strengthens the hypothesis that rapsyn does contain TPRs that mediate self-association. Furthermore, rapsyn self-association requires at least two TPRs. While rapsyn-(1-90) was sufficient for self-association, rapsyn constructs consisting of N-myristoylated TPR1 (rapsyn-(1-41)) or TPR2 (rapsyn-(1-15, 33-90)) alone were insufficient for self-association. Since rapsyn-(1-15, 91-287)-GFP, containing only TPRs 3-7, was also capable of clustering, it is clear that other TPRs can substitute for TPRs 1 and 2. The results concerning the interactions of rapsyn structural domains with wild-type rapsyn provide further evidence that the TPR motifs mediate rapsyn self-association. Rapsyn-(1-15)-GFP, containing the consensus sequence for N-myristoylation, was targeted to the plasma membrane but it did not associate with clustered, full-length rapsyn. However, rapsyn-(1-90)-GFP was co-localized with clustered full-length rapsyn. In contrast, rapsyn-(287-340)-GFP (coiled-coil domain) and rapsyn-(351-411)-GFP (RING-H2 domain) each failed to associate with rapsyn clusters.
TPR domains have been widely recognized as protein interaction domains
(reviewed in Ref. 31 and 32). Identified TPR binding motifs include
short peptide sequences or helix bundles. Within protein chaperone
complexes, the EEVD sequences at the COOH termini of Hsp70 and Hsp90
bind to distinct TPRs in the NH2- and COOH-terminal domains
of Hop (33). In a yeast transcription repressor complex, TPRs 1-3
within SSN6 can bind to a four helix bundle at the NH2 terminus of Tup1 (34) or to a tripeptide sequence within the homeo
domain of a cell type regulator, 2 (35). However, a TPR domain can
also be involved in more distributed binding interactions. Amino acid
side chains distributed in 7 TPRs in the
-subunit of protein
prenyltransferase make extensive contacts with the
-subunit side
chains (36), and based upon yeast two-hybrid and biochemical assays,
the TPR domain of the serine/threonine phosphatase protein 5 is
involved in binding to the TPR domains of the CDC16 or CDC27 subunits
of the anaphase promoting complex (37), but not to PEX5, an unrelated
TPR protein. Since there are no previous reports of TPR domains
mediating protein self-association, it will be of particular interest
to define the structure of the rapsyn-(1-90), the minimal
self-association domain, and also to determine whether the rapsyn TPR
domain is involved in rapsyn binding to other proteins.
Rapsyn TPRs and nAChR Clustering-- Analysis of rapsyn mutants containing TPR deletions revealed that nAChR clustering was still seen for mutant rapsyns lacking TPRs 3-6 and, furthermore, that deletions of TPRs 1 and 2 or TPR 7 (proximal to the coiled-coil domain) were tolerated. Changes in the relative orientation of rapsyn's coiled-coil domain or in the distance between the membrane anchoring site (N-Myr) and the coiled-coil domain of rapsyn due to internal TPR deletions may affect the ability of rapsyn to interact with nAChRs efficiently. Alternatively, it may be that for these deletion mutants the ability to cluster nAChRs might be particularly sensitive to the levels of protein expression or turnover.
Properties of the RING-H2 Domain-- Although nonmyristoylated rapsyn (20) or rapsyn-GFP (6) is targeted to the nucleus, the primary structure of rapsyn does not contain any known consensus sequence for nuclear localization. However, the cysteine-rich domain of rapsyn is a RING-H2 domain, a structural motif found in many transcription factors localized in the nucleus (15). Hence, we reasoned that in the absence of a dominant membrane-targeting signal such as N-Myr, the RING-H2 domain of rapsyn might contribute to its nuclear retention. Consistent with this, rapsyn-(G2A-360), which lacks the RING-H2 domain, was found in the cytosol and was excluded from the nucleus. Thus, with the myristoylated NH2-terminal domain of rapsyn acting as dominant membrane targeting signal, the RING-H2 domain can potentially interact with other proteins at the cell surface. In the absence of the membrane-targeting signal, rapsyn's RING-H2 domain may interact with proteins that are normally localized to the nucleus and by virtue of this interaction be targeted to the nucleus.
An Homology Model of the Rapsyn TPR Domain--
To provide a
structural rationale for rapsyn activity, we constructed a model of
rapsyn (Fig. 9A) based upon
the three-dimensional structure of the 3 TPRs in the serine/threonine
protein phosphatase 5 (17). Rapsyn amino acid sequence 6-279 was
modeled as having 7 TPRs (14). Rapsyn amino acids 298-331 was modeled
as a single -helix, based on our previous results (6) indicating
that rapsyn-(298-331) formed a helix. TPRs 1-7 (rapsyn-(1-287)) form a super-helix extending ~70 Å in length. The concave (inner) surface of the superhelix has a diameter of ~25 Å, while that of the
external surface is ~40 Å. In this context, it is interesting to
note that in the Torpedo postsynaptic membrane, the diameter
of the cytoplasmic projection is ~65 Å (38). In our model, helix B
of TPR 7 ends at rapsyn-(279), and rapsyn-(286-331) is modeled as a
single helix beginning with three turns (rapsyn-(286-297)) packed
along helix B of TPR7 that precede the predicted coiled-coil domain
(rapsyn-(298-331)). With this packing the helix extends ~70 Å beyond the TPR domain with the hydrophobic surface of the coiled-coil
domain oriented toward the concave surface of the TPR superhelix. We
should emphasize that we have no experimental data that constrain the
orientation of the coiled-coil domain relative to the TPR domain. An
interesting alternative packing would be for the coiled-coil domain of
rapsyn (potentially in association with a coiled-coil domain from nAChR subunit(s)) to be actually encompassed within the TPR superhelix. Such
a structure has been proposed for a yeast transcription repressor complex, where a four-helix bundle at the NH2 terminus of
Tup1 is bound within the superhelix groove of TPRs 1-3 of SSN6
(34).
|
Fig. 9a is a surface representation of rapsyn-(1-340) with acidic side chains in blue and basic in red. The two images rotated by 180° are presented to emphasize the surface structure, and the arrows point to the interior (concave) surface of the superhelix at the level of TPR2 (left) and TPR7 (right). Except for Glu83 and Arg90 (in TPR3) and Asp254 (in TPR7), whose side chains point directly into the groove of the superhelix, all the other charged residues (18 Glu, 12 Asp, 18 Lys, and 14 Arg) within rapsyn-(1-287) are located either on the outer surface or in the loops connecting the TPR domains, often as ion pairs. Among these charges, it is striking that there are five noncontiguous lysines at positions 6, 11, 23, 30, and 34 in TPR1 on the external surface (Fig. 9b). Contiguous stretches of positively charged residues have been shown to be required for the membrane association of other myristoylated (src and MARCKS) or palmitoylated (GAP43) proteins (reviewed in Ref. 39). Because rapsyn primary structure does not have such a contiguous stretch of positively charged residues, it is possible that the three-dimensional alignment of these lysine residues close to the myristoylated amino terminus plays the equivalent role in rapsyn's plasma membrane association.
Based on the primary structure it has been suggested (40) that
rapsyn-(82-110), which contains a heptad repeat of Leu/Ile residues,
would be organized as an helix and function as a leucine zipper
coiled-coil motif potentially important for nAChR clustering (20).
However, in our model, this sequence is within TPR3, with the leucines
distributed on the two helices and contributing to the helix packing.
Our data clearly demonstrate that rapsyn self-association is mediated
by the TPR domain. It remains to be determined whether this
self-association is mediated by interactions involving the loops
between the TPRs or either the external or the internal surfaces of the
superhelix. While there has been no evidence of self-association in the
known TPR structures, early studies of the TPR domain of the yeast
nuc 2+ protein, a truncated polypeptide with
nine tandem TPRs, provided evidence that aggregates of as many as
several hundred molecules could be formed (41). The crystal structure
of the armadillo (arm) repeat domain of the nuclear import factor
karyopherin , consisting of 10 repeats of a three-helix bundle,
reveals a homodimer with extensive contacts within the groove (42).
In the present study, we have provided evidence that the rapsyn
coiled-coil domain is essential for nAChR clustering and at least two
TPR repeats are necessary and sufficient for rapsyn self-association.
nAChR clustering is retained for rapsyn constructs that contained the
coiled-coil domain and as few as three TPRs. In the future, it will be
of interest to identify the nAChR domain(s) involved in interaction
with rapsyn and to determine the structure of the rapsyn coiled-coil
domain in association with the nAChR domains.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants NS 19522 and NS 18458 and by a Howard Hughes Medical Institute Predoctoral Fellowship (to M. J. B.).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.
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, November 21, 2000, DOI 10.1074/jbc.M009888200
2 J. Cohen, unpublished data.
3 J. Cohen, unpublished observations.
![]() |
ABBREVIATIONS |
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The abbreviations used are: nAChR, nicotinic acetylcholine receptor; GFP, green fluorescent protein; TPR, tetratricopeptide repeat; PCR, polymerase chain reaction.
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