Acetylcholine Receptor Alpha-subunit Research Articles

Phosphorylation of myogenin in chick myotubes: regulation by electrical activity and by protein kinase C. Implications for acetylcholine receptor gene expression.

We have analyzed the potential role of myogenin in the regulation by electrical activity of the expression of the acetylcholine receptor (AChR) alpha-subunit gene in cultured chick embryonic myotubes. The state of phosphorylation of myogenin was followed by 32P-labeling and immunoprecipitation with an anti-myogenin antibody. In electrically active myotubes myogenin is phosphorylated, while it is dephosphorylated in electrically silent myotubes following tetrodotoxin (TTX) treatment. Accordingly, nuclear protein kinase C (PKC) activity decreases in TTX-treated myotubes. Myogenin dephosphorylation is also observed upon incubation of myotubes with GF109203X, a pharmacological agent which specifically inhibits PKC activity. Both treatments cause similar increases in the expression of the AChR protein. The effects are not additive. Thus TTX and GF109203X most probably affect a common process. Recombinant chick myogenin binds to myogenic sites (E boxes) present in the AChR alpha-subunit promoter but loses this binding capacity after phosphorylation. As a working hypothesis we propose that repression of AChR biosynthesis by electrical activity results, at least partly, from phosphorylation of myogenin via the PKC pathway.

Conserved tyrosines in the alpha subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists.

Studies with site-directed labeling reagents have identified residues near the ligand binding pocket of the nicotinic acetylcholine receptor. Among these residues are three conserved tyrosines, Tyr-93, Tyr-190, and Tyr-198 of the alpha subunit. Previous studies combined mutagenesis, expression in Xenopus oocytes, and dose-response analysis to examine contributions of these tyrosines to agonist affinity. In this study, we prepared a series of mutants at each position, expressed them in 293 HEK cells, and studied binding of agonists and antagonists to mutant receptors on intact cells. We show that all three tyrosines contribute to binding of agonists, and that each tyrosine contributes roughly equally to the binding energy. Although the contributions are roughly equivalent, the nature of the contribution is not equivalent at each position. For Tyr-93 and Tyr-190 the aromatic hydroxyl is essential, whereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand tetramethylammonium, indicating that these tyrosines contribute to stabilization of the quaternary ammonium portion of agonist. Tyr-190 and Tyr-198 also contribute to binding of the competitive antagonist dimethyl-d-tubocurarine; the side chain specificity for binding supports tyrosine interactions with one of two quaternary ammonium groups in dimethyl-d-tubocurarine. Y190F, in addition to altering binding affinity, also affects the equilibrium between activatable and desensitized receptor states.

Interaction of protein ligands with receptor fragments. On the residues of curaremimetic toxins that recognize fragments 128-142 and 185-199 of the alpha-subunit of the nicotinic acetylcholine receptor.

Using a solid-phase assay, we found that 3H-labeled alpha Cobtx from Naja naja siamensis, a long-chain curaremimetic toxin, and 3H-labelled toxin alpha from Naja nigricollis, a short-chain toxin both bind specifically but with substantially different affinities (Kd = 4 x 10(-7) M and 50 x 10(-6) M) to fragment 185-199 (T alpha 185-199) of the alpha-subunit of the acetylcholine receptor (AcChoR) from Torpedo marmorata. Then we show that monoderivatizations of residues common to both long-chain and short-chain toxins (Tyr-25, Lys-27, Trp-29, and Lys-53) or to long-chain toxins only (Cys-30 and Cys-34) do not affect the binding of the toxins to T alpha 185-199, suggesting that none of these invariant residues in implicated in the recognition of this AcChoR region. alpha Cobtx and toxin alpha bind to the fragment 128-142 (T alpha 128-142) with more similar affinities (Kd = 3 x 10(-7) M and 1.4 x 10(-6) M) and their binding is dramatically affected by the single abolition of the positive charge of Lys-53, an invariant residue that contributes to AcChoR recognition. Therefore, the data indicate that Lys-53 more specifically recognizes the 128-142 region of AcChoR. Other monoderivatizations have no effect on toxin binding. The approach described in this paper may be of great help to identify toxin residues that establish direct contact with receptor fragments.

Calcium influx blocks the skeletal muscle acetylcholine receptor alpha-subunit gene in vivo.

The transcriptional activity of the acetylcholine receptor alpha-subunit gene was measured in denervated chick skeletal muscle in response to calcium-active drugs, using a ribonuclease protection version of the conventional run-off assay. The L-channel agonist (-)Bay-K6844 and the calcium ionophore A23187 mimicked, and the intracellular chelator BAPTA and the calcium channel blockers D600 and nifedipine blocked, the effect of electrostimulation. These results suggest that influx of extracellular calcium is an integral component of the membrane depolarization-receptor gene inactivation cascade.

Myogenin and acetylcholine receptor alpha gene promoters mediate transcriptional regulation in response to motor innervation.

Several genes expressed in skeletal muscle are transcriptionally repressed by electrical activity arising from motor innervation and are rapidly induced following denervation. Among these are genes encoding the subunits of the nicotinic acetylcholine receptor (AChR) and the myogenic helix-loop-helix protein myogenin, which activates muscle-specific genes. To understand how electrical activity arising from motor innervation is converted into a transcriptional response, we have attempted to localize cis-acting sequences in the AChR alpha subunit and myogenin genes sufficient to direct activity-dependent transcription. Here we show that an 111-base pair and a 335-base pair region from the promoters of the AChR alpha subunit and myogenin genes, respectively, can confer activity-dependent regulation to a linked reporter gene in transgenic mice. The presence of binding sites for myogenic helix-loop-helix proteins in both of these regulatory regions is consistent with the hypothesis that these myogenic regulators serve as nuclear targets for the signaling cascade through which motor innervation leads to changes in gene transcription in skeletal muscle.

Mapping of the polypeptide chain organization of the main extracellular domain of the alpha-subunit in membrane-bound acetylcholine receptor by antipeptide antibodies spanning the entire domain.

To study the organization of the polypeptide chain of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha-subunit, we examined the ability of the native membrane-bound AChR of Torpedo californica (T-AChR) to bind a panel of antibodies against overlapping synthetic peptides which collectively encompassed this entire domain. Antibodies against the alpha-chain peptides alpha 1-16, alpha 89-104 and alpha 158-174 were able to bind to membrane-bound T-AChR. Other anti-peptide antibodies showed little or no binding to T-AChR in the membrane. It is concluded that regions alpha 1-16, alpha 89-104 and alpha 158-174 are highly exposed on the surface of the alpha subunit of membrane-bound AChR.

Processing requirements of two acetylcholine receptor derived peptides for binding to antigen presenting cells and stimulation of murine T cell lines.

We have previously identified two myasthenogenic T cell epitopes of the human acetylcholine receptor (AChR) alpha subunit, peptides p195-212 and p259-271. These peptides were the immunodominant T cell epitopes of AChR in SJL and BALB/c mice respectively, and only cryptic in C3H.SW strain. In order to find out whether these mouse strains differ in their requirements for processing of the same T cell epitopes, we used p195-212 specific T cell lines from SJL, TCSJL195-212, and C3H.SW, TCSW195-212, mice, and p259-271 specific T cell lines from BALB/c, TCBALB/c259-271, and C3H.SW, TCSW259-271, mice. The peptide-specific proliferative responses of the lines TCSW195-212 and TCSW259-271, originated from strains in which these peptides are cryptic epitopes, were inhibited significantly in the presence of several inhibitors of proteases or glutaraldehyde-fixed antigen presenting cells (APC). In contrast, the proliferative responses of the lines TCSJL195-212 and TCBALB/c259-271, established from strains in which these peptides are immunodominant, were only slightly affected by the above inhibitors or by fixation of the APC. Using a direct binding assay of biotinylated peptides to live intact APC, we showed that peptides p195-212 and p259-271 preserved their binding capacity to APC of SJL and BALB/c mice respectively when processing was inhibited. Thus, the AChR peptides that represent cryptic T cell epitopes have to be processed before they can be recognized by T cells, whereas no further processing is necessary for APC presentation and T cell stimulation when these peptides are immunodominant epitopes.

ALS and SAD-like nicotinic acetylcholine receptor subunit genes are widely distributed in insects.

Segments of nicotinic acetylcholine receptor alpha subunit genes have been isolated from a panel of insect species by polymerase chain reaction, using degenerate oligonucleotide primers designed to recognize conserved regions of the Drosophila melanogaster ALS and SAD genes. The amplified segments encode elements of typical alpha-subunits anticipated to play roles in ligand binding and ion channel formation. Each is also clearly either ALS or SAD-like. The predicted protein sequences display extremely high levels of conservation (over 85% for each subtype) even though derived from very distantly related insect species.

Increased gene expression of acetylcholine receptor and myogenic factors in passively transferred experimental autoimmune myasthenia gravis.

The passive transfer of myasthenia gravis by injection of mAb against muscle acetylcholine receptor (AChR) alpha-subunit, results in increased expression of AChR subunit genes, mainly at synaptic regions. The gene expression of AChR and of other muscle-specific proteins is regulated in a similar manner in passively transferred experimental autoimmune myasthenia gravis (EAMG) and in AChR-induced EAMG. Administration of AChR-specific mAb leads to a significant reduction in muscle AChR content and to an elevation in the mRNA levels corresponding to the adult, synaptic type of the receptor, as shown by Northern blot and in situ hybridization analyses. The mRNA levels of the myogenic factors myogenin and MRF4 are also increased moderately, whereas MyoD transcript levels remain unchanged. Thus, passive transfer of EAMG by mAb directed to defined epitopes of AChR alpha-subunit provides a suitable model for analyzing and following the cascade of molecular events triggered by anti-AChR immunopathologic antibodies and may shed light on the regulatory mechanisms underlying the human disease as well.

Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable.

Acetylcholine receptor subunit mRNA changes in burns are different from those seen after denervation: the 1993 Lindberg Award.

Neuromuscular dysfunction of burn trauma is evidenced as muscle weakness and altered sensitivity to neuromuscular relaxants. A biochemical characteristic of the neuromuscular dysfunction is the proliferation and spread of acetylcholine receptors (AChR) throughout the skeletal muscle membrane. Depending on whether the neuromuscular dysfunction is presynaptic, synaptic, or postsynaptic in origin, the transcripts that induce the proliferation of AChR differ. This study, by quantitation of mRNA transcripts of AChR in muscle, attempts to characterize the cause of the neuromuscular dysfunction of burn trauma. Examination of the levels of mRNA encoding alpha, beta, epsilon, gamma, and delta subunits of AChR with northern blot analysis indicate significant (p < 0.03) elevations of beta-subunit mRNA with a trend for increased levels of alpha and delta transcripts. An increase of gamma-subunit mRNA, typical of presynaptic or nerve-mediated neuromuscular dysfunction, was not observed after burns. That neuronal (presynaptic) factors do not cause the neuromuscular dysfunction was confirmed by the lack of elevation of transcripts of myoD and myogenin, which also increase in "denervation states." These findings indicate that the neuromuscular dysfunction of burn trauma is most likely related to synaptic or postsynaptic factors. Further characterization of the cause of the synaptic and postsynaptic neuromuscular changes in burns will have implications for the choice of therapeutic agents to rectify the neuromuscular dysfunction.

NMR solution structure of an alpha-bungarotoxin/nicotinic receptor peptide complex.

We report the two-dimensional nuclear magnetic resonance (NMR) characterization of the stoichiometric complex formed between the snake venom-derived long alpha-neurotoxin, alpha-bungarotoxin (BGTX), and a synthetic dodecapeptide (alpha 185-196) corresponding to a functionally important region on the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) obtained from Torpedo californica electric organ tissue. BGTX has been widely used as the classic nicotinic competitive antagonist for the skeletal muscle type of nAChR which is found in the avian, amphibian, and mammalian neuromuscular junction. The receptor dodecapeptide (alpha 185-196) binds BGTX with micromolar affinity and has been shown to represent the major determinant of BGTX binding to the isolated alpha-subunit. Previous studies involving covalent modification of the native nAChR from Torpedo membranes with a variety of affinity reagents indicate that several residues contained within the dodecapeptide sequence (namely, Tyr-190, Cys-192, and Cys-193) apparently contribute directly to the formation of the cholinergic ligand binding site. The NMR-derived solution structure of the BGTX/receptor peptide complex defines a relatively extended conformation for a major segment of the "bound" dodecapeptide. These structural studies also reveal a previously unpredicted receptor binding cleft within BGTX and suggest that BGTX undergoes a conformational change upon peptide binding. If, as we hypothesize, the identified intermolecular contacts in the BGTX/receptor peptide complex describe a portion of the contact zone between BGTX and native receptor, then the structural data would suggest that alpha-subunit residues 186-190 are on the extracellular surface of the receptor.

Xenopus muscle acetylcholine receptor alpha subunits bind ligands with different affinities.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentameric complex composed of two identical alpha (the ligand-binding subunit) and one each of beta, gamma, and delta subunits. All muscle-like alpha subunits appear to be encoded by a single gene except for Xenopus where two genes have been identified (X alpha 1a and X alpha 1b). By establishing stable cell lines expressing each Xenopus alpha subunit plus Torpedo beta gamma delta (XaT and XbT), we were able to investigate immunological and ligand-binding properties of these two Xenopus subunits. We determined that each subunit was capable of proper assembly with beta gamma delta subunits, each hybrid AChR cell line responded to bath-applied acetylcholine as determined by 22Na+ influx, but the two hybrid AChRs had distinct ligand-binding properties. The most striking difference between the two hybrids was observed with alpha-bungarotoxin (BuTx) binding. The KD for BuTx binding to XbT hybrids was virtually identical to that of Torpedo and most other muscle AChRs, approximately 10(-10)-10(-11) M. The KD for BuTx binding to XaT hybrids, however, was approximately 1000-fold weaker (6 x 10(-8) M). This weak binding was due primarily to a very rapid BuTx off-rate (4.4 x 10(-2) s-1 for XaT AChRs compared with approximately 1.6 x 10(-5) s-1 for all-Torpedo and XbT AChRs).

Inhibition of T-cell reactivity to myasthenogenic epitopes of the human acetylcholine receptor by synthetic analogs.

The synthetic peptides p195-212 and p259-271, representing amino acids 195-212 and 259-271 of the alpha subunit of the human acetylcholine receptor, preferentially stimulate T cells of patients with myasthenia gravis and are immunodominant T-cell epitopes in SJL and BALB/c mice, respectively. We designed and synthesized analogs of these peptides that contain single amino acid substitutions. An analog of peptide p195-212, no. 455 (Met-207-->Ala), was capable of inhibiting up to 100% of the proliferative responses of a p195-212-specific T-cell line originating from the high-responder strain SJL. Similarly, an analog of p259-271, no. 306 (Glu-262-->Lys), was capable of inhibiting up to 93% of the proliferative responses of the p259-271-specific T-cell line originating from high-responder BALB/c mice. Analog 306 also inhibited up to 43% of the proliferative responses of p259-271-primed lymph node cells in an in vitro proliferation assay. To test the in vivo inhibitory activity of the analogs, mice were primed with the myasthenogenic peptides in complete Freund's adjuvant concomitant with administration of the analogs in aqueous solution. Administration of analogs 455 and 306 led to decreased proliferative responses of up to 70% by peptide p199-212-primed lymph node cells and up to 85% by peptide p259-271-primed lymph node cells. Similar results were obtained whether the analogs were administered i.v. or i.p. Thus, these analogs are good candidates for specific immunomodulatory therapy for patients with myasthenia gravis.

Purification of the bovine nicotinic acetylcholine receptor alpha-subunit expressed in baculovirus-infected insect cells.

The bovine skeletal muscle nicotinic acetylcholine receptor alpha-subunit was produced in insect, Spodoptera frugiperda (Sf9), cells by infection with a recombinant baculovirus. The expressed alpha-subunit protein could not be solubilized efficiently with Triton X-100 or sodium cholate, but could be solubilized efficiently with Zwittergent 3-14, sodium dodecyl sulfate or tris(hydroxymethyl)aminomethane dodecylsulfate. After solubilization of the alpha-subunit protein with Zwittergent 3-14 from the Triton X-100-insoluble fraction, the alpha-subunit protein was purified by concanavalin A-Sepharose chromatography and DEAE ion-exchange chromatography. A milligram quantity of the alpha-subunit protein could be purified from 8 g (wet weight) of Sf9 cells infected with the recombinant baculovirus. Chromatographic analyses including hydroxyapatite chromatography, DEAE ion-exchange chromatography, gel filtration chromatography and chromatofocusing and sucrose density gradient centrifugation analysis suggest that the purified alpha-subunit protein is homogeneous. The purified alpha-subunit protein had a high affinity for 125I-alpha-bungarotoxin and was glycosylated with a high mannose-type N-linked oligosaccharide side chain. These results indicate that purification of ion channel proteins produced by the baculovirus expression system is a promising approach to structural analysis of ion channel proteins, which are extremely rare membrane proteins in native tissues.

Differential recognition by T cells of the P3A+ and P3A- isoforms of the human acetylcholine receptor alpha-subunit.

Differential recognition by T cells of the P3A+ and P3A- isoforms of the human acetylcholine receptor alpha-subunit.

Nine residues influence the binding of alpha-bungarotoxin in alpha-subunit region 185-200 of human muscle acetylcholine receptor.

Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom alpha-neurotoxin antagonists of acetylcholine [e.g., alpha-bungarotoxin (alpha-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR alpha-subunit region 177-208, we previously localized a pharmacologically specific binding site for alpha-BTx in segment 185-199. To define in more detail the residues that influence the binding of alpha-BTx to this region, we prepared 16 peptide analogues of the alpha-subunit segment 185-200, with the amino acid L-alanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro194 was substituted with alanine. This implies that any change in alpha-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence alpha 185-200 in solution-phase competition with native human AChR for binding of 125I-labeled alpha-BTx. The binding of alpha-BTx by analogue peptides with alanine substituted for Tyr190, Cys192, or Cys193 was greatly diminished. Binding of alpha-BTx to peptides containing alanine replacements at Val188, Thr189, Pro194, Asp195, or Tyr198 was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser191 significantly increased alpha-BTx binding (p < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen.

Muscle-specific expression of the acetylcholine receptor alpha-subunit gene requires both positive and negative interactions between myogenic factors, Sp1 and GBF factors.

The dependence of the muscle-specific enhancer of the acetylcholine receptor alpha-subunit gene on other domains of the promoter has been analysed by performing point mutagenesis and modular reconstitution of the enhancer--promoter sequences. The enhancer is inactive in the absence of the proximal region containing an Sp1 binding site and an overlapping G-C homopolymer binding factor site (referred to as GBF). The proximal region can be replaced by an Sp1 binding site from SV40 or an MEF-2 binding site from the muscle creatine kinase gene. Specific mutation of the Sp1 site markedly affects transactivation by CMD1 or myogenin. Mutation of the GBF binding site leads to higher promoter activity in primary cultures of chick myotubes or in quail fibroblasts. In addition, binding of a purified Sp1 protein prevents the binding of GBF in vitro. It is proposed that in the case of the alpha-subunit promoter, the myogenic factors activate transcription in cooperation with Sp1, and that GBF contributes to muscle-specific expression of the promoter by interfering with Sp1 binding in nonmuscle muscle cells or myoblasts.

Monoclonal antibodies against synthetic sequences of the nicotinic receptor cross-react fully with the native receptor and reveal the transmembrane disposition of their epitopes

Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100. mAbs against peptide alpha 304-322, alpha 332-350, and alpha 360-378 cross-reacted with most or all Triton-solubilized TAChR molecules and, in immunoelectron microscopy experiments, bound to the cytoplasmic surface of AChR-rich postsynaptic membrane fragments. Two mAbs specific for the sequence alpha 376-393, proposed to form an amphypathic alpha helix possibly involved in formation of the ion channel, recognized only approximately 35% of Triton-solubilized TAChR molecules and did not react with membrane-bound TAChR. All of these sequence-specific antibodies recognized SDS-denatured TAChR alpha subunit in Western blots. MAbs specific for the amino-terminal sequence region of the alpha subunit, alpha 1-20, and for the sequences alpha 291-308, alpha 346-364, and alpha 390-409 did not recognize native TAChR. A mAb directed against the carboxyl-terminal region, alpha 420-437, recognized with low apparent titer Triton-solubilized TAChR, not membrane-bound TAChR. In conclusion, a complex membrane protein, TAChR, contains several continuous sequence segments exposed on the TAChR surface, because different mAbs raised against certain synthetic sequences recognized most or all native TAChR molecules. By analogy, it should be possible for most proteins of known sequence to raise anti-peptide antibodies fully cross-reactive with the native cognate protein.