From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636
The assembly of the four homologous, but distinct,
ARTICLE
Top
Article
References
,
,
, and
subunits of the nicotinic acetylcholine receptor (nAChR) into the pentamer
2
presents a
unique opportunity to delineate the individual amino
acid side chains that contribute to the assembly process, and to examine the pathway responsible for subunit assembly and expression at the cell surface. It is
well established that subunits assemble into the circular
order of
, where the
subunit resides between
the two
subunits and two binding sites are found at
the
and
interfaces (for detailed reviews, see Karlin and Akabas, 1995
; Hucho et al., 1996
). Chirality of
the order of the subunits has also been proposed on
the basis of cross-linking of toxins with known structures to the receptor (Machold et al., 1995
; Utkin et al.,
1997
). The subunits are glycoproteins composed of
~450-520 amino acids that traverse the membrane
four times (Karlin and Akabas, 1995
); the extracellular
domain is formed from the amino terminal 210 residues (Chavez and Hall, 1991
; Fig. 1). Sequence elements in this domain specify both ligand recognition and the arrangement of subunits (Blount and Merlie,
1989
; Sine and Claudio, 1991
; Yu and Hall, 1991
; Verrall and Hall, 1992
; Kreienkamp et al., 1995
). An extended cytoplasmic loop after the third transmembrane domain contains numerous lysine residues (Boulter et al., 1990
) that may also encode signals for the
stability and trafficking of the subunits.
View larger version (33K):
[in a new window]
Fig. 1.
Subunit topology of the homologous acetylcholine receptor subunits. The NH2-terminal extracellular domain constitutes approximately half of the sequence. An extracellular disulfide loop ( Cys128-142) and glycosylation site (
Asn141) are
conserved throughout the family of ligand-gated ion channels,
which include the nicotinic acetylcholine, the
-aminobutyric acid,
the glycine, and the 5-hydroxytryptamine-3 receptors.
A structural feature common to ligand-gated ion channels is the presence of multiple membrane spans extending to large extracellular and cytoplasmic domains. This separates the multisubunit ion channels from the well-studied receptors of the immune system, such as T cell-receptor subunits that traverse the membrane once. Owing to amino acid sequence similarity, hetero-oligomeric subunit composition, and the conserved positioning of disulfide loops and glycosylation sites, assembly and expression pathways should be shared by the family of ligand-gated ion channels. Therefore, the overall characteristics of assembly and expression identified for acetylcholine receptor biogenesis should be applicable to the other less well studied ligand-gated ion channels.
What is the pathway by which the subunits of the
nAChR assemble in the endoplasmic reticulum (ER)
and become expressed at the cell surface? Four major
features in processing of the subunits appear to direct
expression of the ion channel. First, subunits are inserted in the endoplasmic reticulum membrane and
undergo concurrent folding transitions and other posttranslational modifications throughout the assembly
process. Second, unassembled subunits are susceptible
to rapid degradation (Claudio et al., 1989; Blount and
Merlie, 1990
), and association with chaperones and assembly with neighboring subunits both enhance the
stability of the emerging complex (Keller et al., 1996
,
1998
). Third, the subunits are ordered into the pentameric structure to compose the subunit arrangement of
-
-
-
-
(see Hucho et al., 1996
); specific amino
acid residues in the NH2-terminal domain dictate the
assembly order and the insertion of subunits into the
circular arrangement (Gu et al., 1991
; Kreienkamp et al.,
1995
). Fourth, unassembled subunits and assembled intermediates of the pentameric receptor are retained in
the endoplasmic reticulum; assembly of the pentamer
into its circular arrangement is a requirement for export of the subunits to the Golgi, and then to the cell
surface (Gu et al., 1991
).
Amino Acids at the Interfaces between Subunits Direct the Assembly Pathway
As part of a larger investigation into the amino acid determinants governing ligand specificity and structure,
we have employed subunit transfection into null cells
not expressing the receptor to examine subunit assembly and formation of the ligand recognition sites. This
approach was initially employed by Blount and Merlie (1989) to examine the basis of assembly and the nonequivalence of the two binding sites (Sine and Taylor,
1981
) on the mouse receptor. Subsequent studies by Yu
and Hall (1991)
, Verrall and Hall (1992)
, and Gu et al.
(1991)
have shown that the extracellular domain of the
subunits controls the specificity of subunit oligomerization. More recent studies by Wang et al. (1996a
,b) have
shown that the first transmembrane domain is required
to orient the subunits to assemble. These groups were
able to identify specific partnering of the subunits to
form oligomers and deduce a likely sequence for the
assembly of the transfected subunits.
Previous studies have demonstrated that each of the
homologous subunits is encoded by a separate gene
(see Changeux, 1991). A signal peptide of 20 amino acids at the NH2 terminus directs insertion into the endoplasmic reticulum (ER) such that all subunits are initially embedded and localized to the ER membrane
(Anderson and Blobel, 1981
). The newly synthesized
subunits appear to undergo a folding transition before
assembly, which appears to involve disulfide bond formation (Gelman and Prives, 1996
; Fu and Sine, 1996
;
Green and Wanamaker, 1997
). Folding before assembly may be required to expose the appropriate amino
acids for subunit contact, as suggested by the extended
2-h lag period required to detect the assembled pentamer of subunits (Merlie and Lindstrom, 1983
). It can
be assumed that high affinity interactions between appropriate subunit interfaces occur when subunits contact, forming the assembled intermediates in biogenesis. The subunit intermediates continue to fold and
other subunits are added to the newly exposed faces of
the emerging oligomer. Eventually, all subunits are inserted and assembled into the enclosed pentameric arrangement. Chaperone proteins may assist in the assembly process of the receptor by stabilizing the intermediates and/or promoting folding and assembly.
Amino acid residues positioned at homologous sites
in the subunits appear to direct the partnering during
assembly (Kreienkamp et al., 1995; Sugiyama et al.,
1996
). When cells are transfected with cDNA encoding
only
and
subunits, monomers,
dimers, and
tetramers are observed, as identified by density
gradient sedimentation (Blount et al., 1990
; Kreienkamp et al., 1995
; see Fig. 2). The tetramer
is not
a component of the mature receptor and is an outcome
of transfection with only
and
subunits in the absence of
or
subunits. The
subunit does not assemble between the two
subunits and there is a low propensity to form the unique
interface at the nonligand binding face of the two subunits. Thus, only
dimers, but not tetramers, will form upon cotransfection of
and
. By expressing chimeras of
or
subunits, along with
, a region in
responsible for tetramer formation was identified. From site-specific mutagenesis, two lysines at positions 145 and 150, unique
to
and present as neutral residues in
and
, preclude
from associating with the non-ligand-binding faces of the
subunit. Modification of residue 152 in
the
subunit, a region homologous to 143-153 in
influences the assembly of
with
and
with
subunits
to form dimers that associate at the ligand-binding interface (Sugiyama et al., 1996
). These findings suggest
that homologous residues are positioned in the same
coordinate space in each subunit and are likely to be in
similar contact positions in the assembly process. Upon
transfections of individual subunits, well resolved peaks
in the density gradients enable one to identify the individual species from expression of respective cDNAs
(Kreienkamp et al., 1995
; Sugiyama et al., 1996
).
|
Other studies with subunit chimeras between and
subunits and with mutations in the
subunit have identified other regions responsible for assembly (Yu and
Hall, 1994
; Kreienkamp et al., 1995
). Identification of
residues involved in the assembly of subunits and binding of selective ligands, coupled with labeling by site-
directed chemical and antigenic labels, has led to the refinement of a homology model of the structure of the
extracellular domain of the receptor (Tsigelny et al.,
1997
).
By identifying which combinations of subunits form
stable complexes in cells transfected to express oligomeric assemblies of the receptor subunits, Kreienkamp
et al. (1995) and others (Blount and Merlie, 1989
;
Blount et al., 1990
; Saedi et al., 1991
; Gu et al., 1991
)
have proposed an assembly pathway illustrated in Fig.
2. Specific contacts between two subunits tethered to the ER membrane enable the subunits to form
and
dimers, with
being the more stable dimer (Sugiyama et al., 1996
). The
dimer in turn associates
with the
subunit and the
dimer to form the ion
channel pentamer. From this scheme, it is apparent why a sequence in
or
that allows
or
tetramer formation from dimers would be at a homologous position to residues in the
subunit that influences
or
dimer formation.
An alternative pathway was proposed by Green and
colleagues using stably incorporated Torpedo receptor
subunits in mammalian cells to study the assembly steps
(Green and Claudio, 1993; Green and Wanamaker,
1997
, 1998
). By following subunit incorporation with
pulse labeling and coimmunoprecipitation, they propose a more complex assembly scheme. In this pathway, the first recognized intermediate in the assembly
process is a rapidly forming
trimer (Green and
Claudio, 1993
). Subsequently, a
subunit and then an
additional
subunit are added to the complex (Green
and Claudio, 1993
). The model proposed by Green is
based primarily on the identification of subunit combinations in cells grown at 20°C to slow rates of the assembly process, although a similar assembly pathway for
mouse
subunits expressed at 37°C has been described (Green and Claudio, 1993
). In addition to the
order of subunit assembly, another major distinction
between the two schemes is that the
subunit and the
second
subunit insert between subunits into the
emerging receptor complex (Green and Wanamaker,
1998
). In contrast, in Fig. 2 it is assumed that subunits are added to exposed interfaces, and that the ion channel encloses as the last subunit joins the complex.
Why so different assembly pathways? First, dissimilar
assembly schemes were deduced by transfecting and expressing various combinations of receptor subunits in
separate batches of cells grown at 37°C and identifying
the stable assembled intermediates to reconstruct steps
in the assembly process, in comparison with expressing
all subunits simultaneously at 20°C and following subunit incorporation with metabolic labeling and immunoprecipitation. Kinetically rapidly forming intermediates, such as the -
and
-
dimers, may be undetectable by this method. Differences in temperatures
employed to grow cells assembling the receptor and conditions for receptor solubilization may further contribute to discrepancies in detecting subunit intermediates such as the
tetramer. Second, the assembly
pathway for Torpedo
(and mouse
) sequences
employed by Green and Claudio (1993)
and Green and
Wanamaker (1996) may show some different assembly
characteristics than the combination of mouse
sequences employed in our studies, because the amino
acid sequences of the subunits appear to govern the order of assembly (Kreienkamp et al., 1995
).
Subunit Stability, Processing and Degradation
Nascent subunit peptides residing in the ER are subject
to posttranslational modifications, folding, assembly,
and degradation. Although unassembled subunits
are rapidly degraded (Blount and Merlie, 1990
; Claudio et al., 1989
), our recent studies suggest that association with the chaperone protein, calnexin, substantially reduces the degradation (Keller et al., 1996
, 1998
).
Our studies also reveal that the degradative route for
unassembled subunits dissociated from calnexin is the
ubiquitin-proteasome pathway (Keller et al., 1998
).
Calnexin, as a transmembrane spanning protein, has
the capacity to protect the threaded receptor subunits
on the cytoplasmic, transmembrane, and lumenal domains. It displays features of a lectin because it recognizes an oligosaccharide structure of one terminal glucose linked to mannose residues in the chain, an early structural intermediate in the processing pathway of
nascent N-lined oligosaccharides (Zapun et al., 1997).
The alkaloid castanospermine inhibits the processing
enzymes that trim the nascent oligosaccharide into this
structure primarily recognized by calnexin (Helenius et al., 1997
; Trombetta and Helenius, 1998
). Experiments by Chang et al. (1997)
and Keller et al. (1998)
have demonstrated that treatment with castanospermine increases the degradation of the receptor
subunit (see Fig. 3, compare lanes 3 and 4), implying that
calnexin enhances stability of the associated subunit.
The chaperones, ERp57 and calreticulin, which may be
cryptically associated with the receptor subunit (Keller
et al., 1998
), may also contribute to the stabilization. In
agreement with these observations with castanospermine treatment, earlier studies altering oligosaccha- ride expression and processing have also revealed decreased stability of the receptor
subunit (Smith et al.,
1986
; Blount and Merlie, 1990
). Degradation caused by
castanospermine treatment can be inhibited with the
proteasome inhibitor lactacystin (Fig. 3), suggesting
that subunits with weak calnexin association are targeted to proteasomal hydrolysis.
|
By stabilizing the subunit and thereby reducing dislocation into the ubiquitin-proteasome pathway, calnexin facilitates the incorporation of
subunit into the
oligomeric receptor. In contrast to the isolated subunit,
degradation of assembled
subunits is not substantially altered when expressed in the presence of castanospermine, suggesting that, similar to the association with calnexin, assembly of the subunits themselves
also promotes their stabilization. As subunits assemble,
the neighboring subunit assumes the role of a chaperone stabilizing intermediates in the formation of the assembling receptor.
Fig. 4 summarizes our current view of the processes
involving calnexin association, ubiquitination, and subunit assembly in the control of receptor synthesis. Calnexin (Fig. 4, CN) is attached to monomeric subunits
primarily at the terminal glucose (G) residue in the oligosaccharide. Nevertheless, as a transmembrane protein, it might also protect the receptor subunits at the
cytoplasmic, transmembrane, or extracellular surfaces.
Exposed lysine residues (Fig. 4, K) are recognized by
the ubiquitin conjugation machinery, which enables attachment of polyubiquitin chains (UUU) to these sites.
Owing in part to subunit association with calnexin,
which contains an ER retention sequence (Rajagopalan
and Brenner, 1994), and the tendency for dislocation
of polyubiquitin-tagged glycoproteins to the cytoplasm
(Kopito, 1997
; Suzuki et al., 1998
), unassembled subunits are not exported to the Golgi. Instead, detachment of calnexin further targets the unassembled subunit to proteasomal degradation. The assembly of receptor subunits may cover lysine residues at the
interfaces between the subunits, which should occlude
the ubiquitin conjugation machinery. As subunits assemble, ubiquitin tagging should be reduced and the
nascent assembled receptor subunits should become
more stable.
|
Trafficking of the Subunits from the ER to the Golgi and Cell Surface
A question that emerges from these studies is: what regulates sequestering of unassembled subunits and eventual export of the assembled complex to the Golgi and
cell surface? Ubiquitination and calnexin could also
have roles in the trafficking of unassembled subunits to
the Golgi, due to their inhibitory influence on subunit transport into the secretory pathway. When subunits assemble, calnexin dissociates (Keller et al., 1996), and
the tendency of unchaperoned subunits to dislocate
into the degradative pathway should be reduced. Full
assembly and export of the subunits into the secretory
pathway leading to the Golgi should then be favored.
In summary, by transfecting cells with subunit combinations that are components of the receptor, we and others have identified amino acid residues responsible for subunit assembly and cellular factors that assist in the assembly and expression mechanisms. Additionally, by expressing these subunits in mammalian cells under normal physiological conditions, our findings should be representative of nAChR biosynthesis in vivo. The transient transfection system, where mutations affecting receptor subunit associations can be systematically studied, should continue to provide a valuable approach to understanding subunit assembly. Moreover, studies examining the influence of defined cellular proteins such as calnexin on the assembly and expression of the receptor should likewise yield important new findings on receptor synthesis.
Original version received 17 November 1998 and accepted version received 21 December 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1. | Anderson, D.J., and G. Blobel. 1981. In vitro synthesis, glycosylation and membrane insertion of the four subunits of Torpedo acetylcholine receptor. Proc. Natl. Acad. Sci. USA. 78: 5598-5602 [Abstract]. |
2. | Blount, P., M.M. Smith, and J.P. Merlie. 1990. Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts. J. Cell Biol. 111: 2601-2611 [Abstract]. |
3. | Blount, P., and J.P. Merlie. 1989. Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron. 3: 349-457 [Medline]. |
4. | Blount, P., and J.P. Merlie. 1990. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly. J. Cell Biol. 111: 2612-2622 . |
5. |
Boulter, J.,
A. O'Shea,
Greenfield,
R. Duvoisin,
J. Connolly,
E. Wada,
A. Jensen,
P. Gardner,
M. Ballivet,
E. Deneris,
D. McKinnon, et al
.
1990.
![]() ![]() ![]() |
6. |
Chang, W.,
M.S. Gelman, and
J.M. Prives.
1997.
Calnexin-dependent enhancement of nicotinic acetylcholine receptor assembly
and surface expression.
J. Biol. Chem.
272:
28925-28932
|
7. | Changeux, J.P.. 1991. Compartmentalized transcription of acetylcholine receptor genes during motor endplate epigenesis. New Biol 3: 413-429 [Medline]. |
8. |
Chavez, R., and
Z.W. Hall.
1991.
The transmembrane topology of
the amino terminus of the ![]() |
9. | Claudio, T., H.L. Paulson, W.N. Green, A.F. Ross, D.S. Hartman, and D. Hayden. 1989. Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant. J. Cell Biol. 108: 2277-2290 [Abstract]. |
10. |
Fu, D.X., and
S.M. Sine.
1996.
Asymmetric contribution of the conserved disulfide loop to subunit oligomerization and assembly of
the nicotinic acetylcholine receptor.
J. Biol. Chem.
271:
31479-31484
|
11. | Gelman, M.S., and J.M. Prives. 1996. Arrest of subunit folding and assembly of nicotinic acetylcholine receptors in cultured muscle cells by dithiothreitol. J. Biol. Chem. 27: 10709-10714 . |
12. | Green, W.N., and T. Claudio. 1993. Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially. Cell. 74: 57-69 [Medline]. |
13. |
Green, W.N., and
C.P. Wanamaker.
1997.
The role of the cystine
loop in acetylcholine receptor assembly.
J. Biol. Chem.
272:
20945-20953
|
14. |
Green, W.N, and
C.P. Wanamaker.
1998.
Formation of the nicotinic acetylcholine receptor binding sites.
J. Neurosci.
18:
5555-5564
|
15. | Gu, Y., P. Camacho, P. Gardner, and Z.W. Hall. 1991. Identification of two amino acid residues in the epsilon subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells. Neuron. 6: 879-887 [Medline]. |
16. | Helenius, A., E.S. Trombetta, D.N. Herbert, and J.F. Simons. 1997. Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7: 193-200 . |
17. | Hucho, F., V.I. Tsetlin, and J. Machold. 1996. The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor. Eur. J. Biochem. 239: 539-557 [Abstract]. |
18. | Karlin, A., and M. Akabas. 1995. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 15: 1231-1244 [Medline]. |
19. |
Keller, S.H.,
J. Lindstrom, and
P. Taylor.
1996.
Involvement of the
chaperone protein calnexin and the beta-subunit in the assembly
and cell surface expression of the receptor.
J. Biol. Chem.
271:
22871-22877
|
20. |
Keller, S.H.,
J. Lindstrom, and
P. Taylor.
1998.
Inhibition of glucose trimming with castanospermine reduces calnexin association
and promotes proteasome degradation of the alpha-subunit of the
nicotinic acetylcholine receptor.
J. Biol. Chem.
273:
17064-17072
|
21. | Kopito, R.R.. 1997. ER quality control: the cytoplasmic connection. Cell. 88: 427-430 [Medline]. |
22. | Kreienkamp, H.-J., R.K. Maeda, S. Sine, and P. Taylor. 1995. Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor. Neuron. 14: 636-644 . |
23. | Machold, J., C. Weise, Y. Utkin, V. Tsetlin, and F. Hucho. 1995. The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica. Eur. J. Biochem. 234: 423-430 . |
24. | Merlie, J.P., and J. Lindstrom. 1983. Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate. Cell. 34: 747-757 [Medline]. |
25. | Rajagopalan, S., and M.B. Brenner. 1994. Retention of unassembled components of integral membrane proteins by calnexin. Science. 263: 387-390 [Medline]. |
26. | Saedi, M., W.G. Conroy, and J. Lindstrom. 1991. Assembly of Torpedo acetylcholine receptor in Xenopus oocytes. J. Cell Biol 112: 1007-1015 [Abstract]. |
27. |
Sine, S.M., and
T. Claudio.
1991.
Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the
acetylcholine receptor.
J. Biol. Chem.
266:
19369-19377
|
28. |
Sine, S.M., and
P. Taylor.
1981.
Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor.
J. Biol. Chem
256:
6692-6699
|
29. |
Smith, M.M.,
S. Schlesinger,
J. Lindstrom, and
J.P. Merlie.
1986.
The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor.
J. Biol.
Chem.
261:
14825-14832
|
30. | Sugiyama, N., A.E. Boyd, and P. Taylor. 1996. Anionic residue in the alpha-subunit of the nicotinic acetylcholine receptor contributing to subunit assembly and ligand binding. J. Biol. Chem 27: 26575-26581 . |
31. |
Suzuki, T.,
Q. Yan, and
W.J. Lennarz.
1998.
Complex two-way traffic
of molecules across the membrane of the endoplasmic reticulum.
J. Biol. Chem.
273:
10083-10086
|
32. | Trombetta, E.S., and A. Helenius. 1998. Lectins as chaperones in glycoprotein folding. Curr. Opin. Struct. Biol 8: 587-592 [Medline]. |
33. | Tsigelny, I., N. Sugiyama, S.M. Sine, and P. Taylor. 1997. A model of the nicotinic receptor extracellular domain based on sequence identity and residue location. Biophys. J. 73: 52-66 [Abstract]. |
34. |
Wang, Z.Z.,
S.F. Hardy, and
Z.W. Hall.
1996a.
Assembly of the nicotinic acetylcholine receptor. The first transmembrane domains
of truncated alpha and delta subunits are required for heterodimer formation in vivo.
J. Biol. Chem.
271:
27575-27584
|
35. | Wang, Z.Z., S.F. Hardy, and Z.W. Hall. 1996b. Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha-subunit to form a heterodimeric ligand-binding site. J. Cell Biol. 135: 809-817 [Abstract]. |
36. | Utkin, Y.N., A.V. Krivoshein, V.L. Davydov, I.E. Kasheverov, P. Franke, I.V. Maslennikov, A.S. Arseniev, F. Hucho, and V.I. Tseltin. 1997. Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23. Eur. J. Biochem. 253: 229-235 [Abstract]. |
37. | Verrall, S., and Z.W. Hall. 1992. The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell. 68: 23-31 [Medline]. |
38. | Yu, X.M., and Z.W. Hall. 1991. Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor. Nature. 352: 64-67 [Medline]. |
39. | Yu, X.M., and Z.W. Hall. 1994. Amino- and carboxyl-terminal domains specify the identity of the delta subunit in assembly of the mouse muscle nicotinic acetylcholine receptor. Mol. Pharmacol. 46: 964-969 [Abstract]. |
40. | Zapun, A., S.M. Petrescu, P.M. Rudd, R.A. Dwek, D.Y. Thomas, and J.J. Bergeron. 1997. Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. Cell 88: 29-38 [Medline]. |