Nicotinic acetylcholine receptor: Structure, function and main immunogenic region

Advances in Neuroimmunology Vol. 4, pp. 339-354, 1994 Pergamon

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0960-5428(94)00032-8

Nicotinic acetylcholine receptor: function and main immunogenic

Structure, region

Avgi Mamalaki and Socrates J. Tzartos Department

of Biochemistry,

Keywords-Nicotinic antibodies.

Hellenic

acetylcholine

Pasteur receptor,

Institute,

127 Vas. Sofias Ave.,

myasthenia

gravis,

main

Athens

11521, Greece

immunogenic

region,

monoclonal

Introduction The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel gene superfamily, which includes the y-aminobutyric acid (GABA) and glycine receptors (Barnard et al., 1987; Stroud et al., 1990). It contains receptors from the post-synaptic membranes of vertebrate neuromuscular junctions, from the electric organs of electric fish (Torpedo and from the central and Electrophorus) nervous systems of vertebrates and insects. These receptors have several structural and functional features in common, but also have many differences (Lucas and Bencherif, 1992). The muscle-type AChR (i.e. that of the neuromuscular junction and fish electric organs) is the best-known member of the AChR family. It is an integral pentameric membrane protein (Popot and Changeux, 1984) formed from four types of subunits in the stoichiometry @yS or OL$ES (Reynolds and Karlin, 1978; Numa, 1987). Three major factors have facilitated its extensive characterisation: (a) the large quantities that can be isolated from the electric organs of fish, (b) the presence of several snake venom neurotoxins, that bind specifically to the AChR and (c) the application of recombinant DNA techniques. In the pres339

ent review, we will discuss only this type of AChR. Structural and functional studies of the muscle AChR are of great importance as the molecule acts as the autoantigen in the autoimmune disease myasthenia gravis (MG) and in its experimental animal model (Oosterhuis, 1984; Lindstrom et al., 1988; Penn et al., 1992). MG is characterized by weakness and fatiguability of the skeletal muscles. The disease occurs in about 1 in 20,000 people. As the autoantigen is a well characterized molecule, MG represents an excellent model for the study of the molecular interactions involved in a human autoimmune disease.

Function of AChR Synaptic transmission ion channel

-Activation

of the

The muscle AChR plays a critical role in neuromuscular transmission. It binds specifically the neurotransmitter acetylcholine (ACh) and converts rapidly this chemical signal (chemical transmitter) from the neurons into an electrical effect that is propagated along the membrane of the muscle fiber. The AChRs open and close rapidly

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(gating) in response to ACh. Because of the passive flow of cations into and out of the cell (Na+ and K+, respectively), the postsynaptic membrane of the muscle fiber depolarizes, producing the endplate potential. This endplate potential acts on the neighbouring voltage-gated Na+ channels, which open and produce an action potential that is propagated along the muscle fiber surface, leading to contraction (Vincent and Wray, 1992). ACh is released by exocytosis from vesicles situated in the synaptic membrane of the nerve terminal (Heuser et al., 1979). Each vesicle releases a “quantum” of ACh, of about 104 molecules (Kuffler and Yoshikami, 1975), into the synapatic cleft (Del Castillo and Katz, 1957). Normally, 50-3000 vesicles are released at the neuromuscular junction during neuron-muscle stimulation. Two molecules of ACh bind simultaneously to two binding sites present in the extracellular part of the a-subunits of each AChR molecule, which interact in a positively cooperative manner (Changeux, 1990). Cysteines at ~~192 and a193 play a critical role in ACh binding and function (Kao and Karlin, 1986). In addition, residues Tyr93, Trp149, Tyr190 and possibly Tyrl51 and Tyr198 seem to be implicated in ACh binding (Changeux, 1990). The AChR cation channel opens for about one millisecond (msec), allowing up to 5 X lo4 cations to flow through the channel. ACh is rapidly hydrolyzed by acetylcholinesterase, which is located in the synpatic cleft, associated with the basement membrane that surrounds the entire muscle fiber. AChR function has been extensively studied using several agonists and antagonists of ACh and other AChR blockers. Snake venom c-w-toxins are a group of a small proteins of approximate MW 7500-8000 Da, such as a-bungarotoxin, that bind with high affinity (K, approximately 2 X l@llM) to the two a-subunits at or near the ACh binding sites (Maelicke, 1988; Changeux,

1990). Peptides corresponding to residues al70-200 of the AChR a-subunit, are capable of binding cx-bungarotoxin (Neumann et al., 1986; Aronheim et al., 1988; Wilson and Lentz, 1988; Gotti et al., 1988). The smallest c-w-bungarotoxin-binding peptide seems to be a189-195 (Tzartos and Remoundos, 1990); the S-S bond at (-u192193 does not seem to be necessary for binding cY-bungarotoxin (Tzartos and Remoundos, 1990; Griesmann et al., 1990), although it is necessary for ACh binding and function (Kao and Karlin, 1986). Another group of molecules, including local anesthetics, block AChR function noncompetitively; these have been shown to bind within the ion channel (Oberthur et al., 1986; Giraudat ef al., 1987; Changeux, 1990). Modulatory changes of AChR The different functional states of the AChR correspond to different conformational states, as the molecule is a membrane-bound allosteric protein (Changeux, 1981; Lena and Changeux, 1993). Under the action of allosteric regulators, AChR exhibits three functional states: (a) a resting state, in which the AChR is closed and activatable, and has a low affinity for ACh; (b) an active state, in which the AChR is open and (c) a refractory or desensitized state, occurring when the receptor is exposed to a high ligand concentration, in which the AChR is closed and nonactivatable, and with higher affinity for ACh; after the initial depolarization, the activity of the endplate channels diminishes, and the desensitized conformation persists for many seconds before relaxing to the resting state. The resting potential of the muscle plasma membrane is about -70 mV. Neural stimulation by the quanta that are released increases the permeability to cations, producing a near complete depolarization of the endplate. This depolarization generates the

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acetylcholine

action potential. In the absence of stimulation, spontaneous exocytosis of single ACh vesicles occurs, producing a depolarization of about 0.5-1.0 mV (the miniature endplate potential, MEPP) without producing an action potential.

Metabolism

of ACbR

The synthesis and assembly of the AChR subunits have been studied in various systems, such as muscle cell lines, yeast, Xenopus oocytes and fibroblast cell lines (Merlie et al., 1982; Fujita et al., 1986; Mishina et al., 198.5; Blount and Merlie, 1988; Claudio et al., 1987). All these studies have shown that all four subunits are required for the assembly and localization of a fully functional AChR in the surface membrane. Synthesis of each subunit takes place in the rough endoplasmic reticulum (Anderson et al., 1982) and, as in other membrane proteins, the subunits are modified by posttranslational modifications before the AChR is localized in the post-synaptic membrane. This process requires more than 2 hours. After polypeptide synthesis the a-subunits undergo conformational maturation which can be demonstrated using a-bungarotoxin (ol-BuTx) and monoclonal antibody binding tests (Merlie and Lindstrom, 1983; Smith et al., 1987). Conformational changes may be due to disulfide bridging of the two cysteines at positions 128 and 142 of the a-subunit (Blount and Merlie, 1990); this process may be assisted by the BIP binding protein of the endoplasmic reticulum (Blount and Merlie, 1991a; Forsayeth et al., 1992). AChR is assembled in the endoplasmic reticulum (Smith et al., 1987; Gu et al., 1989). Expression studies in eukaryotic cells have shown that a6 and ay or (YE heterodimers are first formed (Blount et al., 1990; Blount and Merlie, 1991b; Gu et al., 1991) by direct interaction of their extracellular

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N-terminal domains (Yu and Hall, 1991; Verrall and Hall, 1992), followed by an association of each heterodimer with the a-subunit. forming the complete AChR molecule. This process means that the two a-subunits have different neighbors. Recently, another model was proposed by Green and Claudio (1993). Using the temperature sensitivity of the Torpedo AChR subunits, they showed that trimerization of (Y, l3 and y subunits occurs within 5 min of synthesis, and that this is followed by the slow addition of the 6 subunit and finally the second (Y subunit joins the complex to form the pentamer. Post-translational modifications of AChR subunits include N-glycosylation and phosphorylation. The N-glycosylations of the polypeptides may contribute to the stability of the subunits and/or to the efficient insertion of the AChR in the plasma membrane (Merlie et al., 1982; Sumikawa and Miledi, 1989; Gehle and Sumikawa, 1991). AChR phosphorylation by at least three kinases seems to be involved in AChR et al., 1991), clustering assembly (Green (Wallace et al., 1991) and function moduet al., 1986; Huganir and lation (Huganir Greengard, 1990; Ferrer-Montiel et al., 1991; Tzartos et al., 1993). All the identified phosphorylation sites (five serines and three tyrosines per Torpedo AChR pentamer) are located in the major cytoplasmic domain of each subunit (Wagner et al., 1991). The pentameric AChR complex is transported to the membrane surface, where it is localized in postsynaptic folds associated with basal lamina and cytoskeleton proteins. The number and distribution of AChR molecules are very critical for neuromuscular transmission. At the mature neuromuscular junctions, the number of packed AChRs is approximately 10,000/~m2 in a region comprising only 0.1% of the total muscle surface. This accumulation at the endplate is mediated by specific factors from the basal lamina and cytoskeleton (Laufer and

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Changeux, 1989; Froehner, 1993) and by diffusible signals from the nerve terminal (Falls et al., 1990; Witzemann et al., 1991; Simon et al., 1992). AChR degradation has been studied in muscle cell cultures using radioactive amino acids and 12sI-labelled a-bungarotoxin. The mechanism involved is endocytosis, which requires energy. In the lysosomes, the AChRs are degraded to the component amino acids, which are released from the cell about 90 min after initiation of internalization. The stability of AChRs is controlled by muscle activity mediated by et al., 1990). The motor neurons (Fumagalli half-life of the embryonic AChRs is about 1 day, while that of the adult AChRs is about et al., 1979). 10-12 days (Steinbach

Structure

of AChR

Biochemical studies have shown that the AChR of skeletal muscle or electric organs is a membrane glycoprotein with a molecular weight of about 290k Da (Maelicke, 1988; Changeux, 1990). The AChRs of skeletal muscle consist of two subtypes, fetal and adult. The adult subtype (cx~B&) is present in adult neuromuscular junctions and it is a high-conductance channel, whereas the fetal subtype (Q3yg) is present in the extrajunctional membrane before innervation (or after denervation) and it is a low-conductance channel (Mishina et al., 1986; Sakmann et al., 1992). The expression of these subtypes is under the control of several neural factors that predominate at different developmental stages (Sakmann et al., 1992). Primary structure of the AChR subunits Recombinant DNA techniques have been used in order to determine the complete cDNA sequences of the (Y- (Noda et al., 1982), B- (Noda et al., 1983b), y- (Claudio et al., 1983) and S-subunits (Noda et al.,

1983b) from Torpedo californica AChR and the cx-subunit from Torpedo marmorata AChR (Devillers-Thiery et al., 1983). Subsequently, DNA probes from the different subunits were used to isolate cDNAs and genomic clones coding for muscle and neuronal AChR subunits (Noda et al., 1983a; Nef et al., 1984, 1988; LaPolla et al., 1984; Boulter et al., 1986; Claudio, 1989). In adult bovine, mouse, rat and human muscle the embryonic mRNA y species is replaced by the homologous adult mRNA coding for the e-subunit (Takai et al., 1985; Numa, 1987; Criado et al., 1988; Buonanno et al., 1989). Comparison of the deduced amino acid sequences for each subunit within a species showed a high degree of homology, with approximately 40% amino acid identity. Even higher degrees of homology are seen in inter-species comparison of a single subunit. Thus, the a-subunit, the most conserved AChR subunit, shows about 80% homology between the human muscle AChR and that of Torpedo electric organ. This homology is not constant along the polypeptide chains. The putative transmembrane regions present the greatest homology (Claudio, 1989). The genes coding for the different subunits evolved from a common ancestral gene by gene duplication (Raftery et al., 1980). Sequence comparison among AChR subunits shows interesting common characteristics. All subunits have a leader peptide in the NH,-terminus of 17-24 amino acids which is processed in the mature protein. The first 210-220 amino acids of the subunits are hydrophilic and represent the main extracellular domain. The C-terminus also seems to be extracellular (Dipaola et al., 1989; Chavez and Hall, 1992). Hydropathy analysis of the deduced amino acid sequences indicated the presence of three hydrophobic domains (Ml, M2 and M3), followed by a long hydrophilic domain and then a fourth hydropobic domain (M4). The long hydrophilic domain is cytoplasmic (Ratnam et al.,

Nicotinic

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1986a; Kordossi and Tzartos, 1987; Chavez and Hall, 1992). The Ml, M2, M3 and M4 domains represent the putative membrane spanning regions (Devillers-Thiery et al., 1983; Noda et al., 1983~; Ratnam et al., 1986a; Lindstrom et al., 1987; Chavez and Hall, 1992; Fig. 1). The hypothesis that the M2 region from each subunit lines the ion channel pore of the AChR was confirmed by various approaches including site-directed mutagenesis (Hucho et al., 1986; Imoto et al., 1988; Leonard et al., 1988). The a-subunit is composed of 437 amino acids. Four cysteine residues form two intrasubunit disulfide bonds in the extracellular domain of the a-subunit. One pair, in positions 128 and 142, seems to be involved in the intracellular retention of most of the assembled AChR complexes (Sumikawa and Gehle, 1992). The second cysteine pair is located at positions 192 and 193, which are known to be near the ACh binding site (Kao et al., 1984; Kao and Karlin, 1986). An

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asparagine-linked glycosylation site occurs at position 141 and its glycosylation seems to be required for the normal maturation of the u-subunit (Blount and Merlie, 1990). Some invariant amino acid residues exist in the Ml-M4 domains of all AChR subunits. Thus, a proline residue is present in the middle of Ml region; another proline residue occurs before the end of the Ml region; two proline residues occur between M2 and M3; a proline residue is found C-terminal to M3; four to five serine and threonine residues occur in M2 and a leucine residue is found in the middle of this domain (Claudio, 1989). Three-dimensional

structure of the AChR

Electron microscopy of tubular crystals of Torpedo post-synaptic membranes has been used to reconstruct the three-dimensional structure of the AChR (Mitra et al., 1989; Toyoshima and Unwin, 1988). At 17A reso-

MIR CHO

Plasma membrane /

Fig. 1. A scheme showing the putative secondary structure of the AChR u-subunit. Four transmembrane helices (Ml, M2, M3, M4) exist in each subunit. The M2 helices from each subunit seem to form the ion channel. The a298-408 region is cytoplasmic. The main loop of MIR is between a67 and 0176. a189-195 binds cu-Bgt; a149 together with algO-198 contribute to the ACh binding site (see text).

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lution, the AChR appears as a cylinder of 12OA in length, with a pseudo-five-fold symmetry axis running down the center of the molecule, and lying approximately perpendicular to the plane of the membrane (Toyoshima and Unwin, 1988, 1990). The three-dimensional structure of the closed configuration of AChR at 9A resolution was recently obtained (Unwin, 1993; Fig. 2). The extracellular part of the molecule has an irregular outer surface, while its inner surface is smooth and forms a wall lining the synaptic entrance of the

channel. This entrance is a tube with a diameter of 20-25A. The synaptic domain of each subunit contains three rods (ahelices), twisted around each other. In the a-subunits, these three helices form a cavity about 3OA above the bilayer surface, which may constitute the ACh-binding site. The membrane-spanning region shows a high degree of rotational symmetry. The central pore is lined by five helices, but is surrounded by a continuous rim corresponding to a P-barrel. Therefore, contrary to what was previously believed, each subunit seems

Fig. 2. Model of the Torpedo AChR. The extracellular parts of the receptor are shown as well as the attached 1993, by permission.)

(S), transmembrane (B) and cytoplasmic (C) cytoplasmic 43K protein (P). (From Unwin,

Nicotinic

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to have only one transmembrane a-helix. The author suggested that this a-helix is the M2 domain, whereas two or three of the Ml, M3, M4 segments of each subunit may form the surrounding P-barrel. The transmembrane M2 helices appear to be kinked and oriented in such a way that their close approach occludes the central pore, forming a barrier to ion permeation.

Antigenic structure of the AChR and its main immunogenic region MG and experimental autoimmune MG are antibody-mediated disorders in which the antigen is the AChR. Animals of various species immunized with intact or fragmented AChR respond with the production of antibodies directed against various sites of the molecule. A single injection of tl kg of purified AChR per rat is sufficient to induce an immune response against the AChR et al., 1976). Generally, when (Lindstrom intact AChR is used as immunogen, most of the antibodies are directed against extracellular parts of the AChR. In contrast, when sodium dodecyl sulfate-denaturated AChR or its isolated subunits are used as immunogens, the majority of the antibodies are directed against cytoplasmic sites of the AChR (Froehner, 1981; Sargent et al., 1984). The majority of rat polyclonal and monoclonal antibodies (mAbs) against intact solubilized AChR from fish electric organ and from human muscle compete for binding to a specific region on each a-subunit, the main immunogenic region (MIR) (Tzartos and Lindstrom, 1980; Tzartos et al., 1981, 1983). Immunization with fetal calf muscle AChR resulted in the production of mAbs against the MIR and also against additional extracellular sites on (Y-, p- and y-subunits (Tzartos et al., 1986). mAbs derived from mice immunized with intact human muscle AChR were found to bind to overlapping regions, including the MIR and the toxin bind-

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ing site (Whiting et al., 1986; Heidenreich et al., 1988). In addition, the majority of serum antibodies from mice immunized with mouse AChR were directed against the MIR (Jermy et al., 1993). Although other sites of the AChR may play an important pathogenic role in MG and experimental autoimmune MG (Marx et al., 1990; Sano et al., 1991; Ashizawa et al., 1992), we intend to review only our current knowledge of the MIR and the anti-MIR mAbs (Tzartos et al., 1991).

Identification

of the MIR

Because of the possible important role of the MIR in the pathogenesis of MG, the identification of this region has been very important (Ratnam et al., 1986; Barkas et al., 1987; Tzartos et al., 1988). The MIR is located extracellularly. This is evident from the fact that anti-MIR mAbs can bind to intact muscle cells in culture (Conti-Tronconi et al., 1981; Tzartos et al., 1985) and are capable of causing AChR loss in experimental rats (Tzartos et al., 1987). Furthermore, the extracellular location of the MIR has been directly visualized in several electron microscopy studies (Swanson et al., 1983; Kubalek et al., 1987). Although binding of anti-MIR antibodies is conformationally-dependent, a weak residual binding of anti-MIR mAbs to denatured AChR subunits and peptides allowed the detailed localization of the MIR. Localization of the MIR to the a-subunit was determined by the binding of several anti-MIR mAbs to the denatured a-subunit (Tzartos and Lindstrom, 1980; Tzartos et al., 1981). Since each AChR molecule has two o-subunits, it also has two MIRs (ContiTronconi et al., 1981). Using several approaches, such as a-subunit proteolytic peptides (Barkas et al., 1986; Ratnam et al., 1986), recombinant a-subunit segments (Barkas et al., 1987) and several synthetic peptides, the MIR was finally localized be-

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tween a61 and a76 of Torpedo and mouse AChR (Barkas et al., 1988) and between ~67 and a76 of human (WNPDDYGGVK) and Torpedo (WNPADYGGIK) AChR (Tzartos et al., 1988, 1990). Human (~67-76 is identical to mouse a67-76, while it differs only by one non-conservative (Asp instead of Ala70) and one conservative (Val instead of Ile7.5) substitution from the equivalent Torpedo decapeptide. The contribution of this region to the MIR was confirmed by site-directed AChR mutants expressed in Xenopus oocytes (Saedi et al., 1990). The epitope of a mouse anti-MIR mAb was also localized within 0165-78 (Wood et al., 1989). The antigenic role of each residue within (~67-76 was tested using peptide analogs of a67-76, in each of which one residue was substituted by alanine (Papadouli et al., 1990), glycine (Bellone et al., 1989) or multiple amino acids (Papadouli et al., 1993). In these studies, the 1~68-71 region was found to be the most critical binding segment of the MIR. The conformation of Torpedo 0167-76 and several of its analogs have been studied by two-dimensional NMR spectroscopy. In DMSO, 0167-76 assumes a folded conformation stabilized by three interactions involving the D71, G74 and K76 amide protons. Any Ala substitution, except that on 1~75, significantly affects the conformation of the decapeptide (Cung et al., 1989). In the presence of anti-MIR mAbs these peptides acquire a folded conformation even in aqueous solutions; an adequate structuration of the N-terminal sequence is required for full molecular recognition by the anti-MIR mAbs, whereas intrinsic nonstructuring of the C-terminal part by Ala substitution is not of primary importance (Cung et al., 1992; Tsikaris et al., 1993). Several approaches have been used to determine the epitope specificities of antibodies in MG sera. Although in some studies synthetic peptides have been used for MG antibody mapping we believe that, since

binding of most anti-AChR antibodies is conformationally-dependent, the outcome of such mapping is misleading. Human MG sera have been tested using competition experiments between anti-MIR mAbs and MG sera for binding to the intact AChR. In these studies, the proportion of serum antibodies inhibited from binding to the AChR by the anti-MIR mAbs varied from patient to patient (from 10 to lOO%), but in the great majority of the sera it was greater than 50% (Tzartos et al., 1982,1985, 1990; Hohlfeld et al., 1987; Heidenreich et al., 1988). Yet, determination of the actual epitope specificities of these antibodies requires the use of direct approaches for the MG antibodies. Use of AChR hybrids and mutants should offer a direct epitope mapping approach. As a first step, Torpedo-mouse AChR hybrids were used to confirm the immunodominance of the a-subunit in MG sera-(Loutrari et al., 1992).

Pathogenicity

of the anti-MIR

antibodies

The potential functional role of the antiMIR mAbs has been investigated in experimental animals and muscle-cell cultures. Five anti-MIR mAbs were capable of inducing MG symptoms in experimental animals within l-3 days. Most of the animals showed severe MG symptoms and lost approximately half of their muscle AChR (Tzartos and Lindstrom, 1980; Tzartos et al., 1987). Similarly, animals injected with two apparently anti-MIR mAbs (Lennon and Griesmann, 1989) also exhibited MG symptoms and loss of muscle AChR (Lennon and Lambert, 1980). In contrast, four mAbs to non-MIR sites did not cause either symptoms of MG or AChR loss (Tzartos et al., 1987). The ability of the anti-MIR mAbs to cause AChR loss via antigenic modulation was tested on fetal calf muscle cells (ContiTronconi et al., 1981), on the mouse muscle cell line BC3Hl (Tzartos et al., 1985),

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on the human rhabdomyosarcoma cell line TE671 (Sophianos and Tzartos, 1989) and on AChR-transfected fibroblast cell lines (Loutrari et al., 1992). Almost all anti-MIR mAbs tested accelerated AChR degradation 2-3 fold, as compared with its degradation rate in the absence of antibody, i.e. they produced an effect very similar to that of the MG serum antibodies (Conti-Tronconi et al., 1981; Tzartos et d., 1985; Tzartos and Starzinski-Powitz, 1986). In contrast, five other mAbs, four of which are probably directed against the cytoplasmic side of the AChR, did not cause antigenic modulation (Tzartos et al., 1985; Tzartos and StarzinskiPowitz, 1986; Sophianos and Tzartos, 1989). The modulatory activity of the anti-MIR antibodies from MG sera has been tested only indirectly, as this antibody fraction cannot be yet isolated quantitatively. The approach used was to shield the MIR with a univalent antibody fragment (Fab or Fv) of an anti-AChR mAb and then to add the MG serum in order to measure its capacity to downregulate the protected AChR (univalent Fab or recombinant single chain Fv antibody fragments do not cross-link by themselves the AChR molecules present in cell cultures and thus they do not cause antigenic modulation). It was found that the AChR in cell cultures is efficiently protected by antiMIR mAb fragments (by 60-70%) against the antigenic modulation induced by human MG sera (Tzartos et al., 1985; Sophianos and Tzartos, 1989; Mamalaki et al., 1993). Thus, the MIR may be the major pathogenic region of the AChR. A scheme for the efficient protection of the MIR in human MG patients may provide a useful thera: peutic approach. However, the application of such an approach would require a number of modifications of the available anti-MIR recombinant antibody fragment (Mamalaki et al., 1993). In conclusion, the study of the MIR offers just one example among many, which emphasize that progress towards understanding

receptor

and treating MG is heavily dependent a detailed knowledge of the structure function of muscle AChR.

347 on and

Acknowledgements We thank Drs T. Barkas, H. Loutrari, R. Matsas, E. Patsavoudi and K. Soteriadou for valuable suggestions. Work in the authors’ laboratory was supported in part by grants from Association Franqaise contre les Myopathies (AFM) and United Nations Industrial Development Organization (UNIDO).

References Anderson, D. J., Walter, P. and Blobel, G. (1982). Signal recognition protein is required for the integration of acetylcholine receptor 6 subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membranes. J. Cell Biol. 93:501-506.

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Aschizawa, T., Ruan, K.-E., Jinnai, K. and Atassi, M. Z. (1992). Profile of the regions on the a-chain of human acetylcholine receptor recognized by autoantibodies in myasthenia gravis. Mol. Immunol. 29:1507-1514. Barkas, T., Gabriel, J. M., Juillerat, M., Kokla, A. and Tzartos, S. J. (1986). Localization of the main immunogenic region of the nicotinic acetylcholine receptor. FEBS Lett. 196:237-241.

Barkas, T., Mauron, A., Roth, B., Alliod, C., Tzartos, S. J. and Ballivet, M. (1987). Mapping the main immunogenic region and toxin binding site of the nicotinic acetylcholine receptor. Science 235:77-80. Barkas, T., Gabriel, J.-M., Mauron, A., Hughes, G. J., Roth, B., Alliod, C., Tzartos, S. J. and Ballivet, M. (1988). Fine localisation of the main immunogenic region of the nicotinic acetylcholine receptor to residues 61-76 of the a-subunit. J. Biol. Chem. 263:591&5920.

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