Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.     

Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles

We tested the effects of 62 monoclonal antibodies (mAbs) to acetylcholine receptors from Torpedo californica on the function of receptor reconstituted into lipid vesicles. Two of these mAbs, mAbs 148 and 168, inhibited carbamylcholine-induced 22Na+ uptake into vesicles. The rate of 125I-labeled alpha-bungarotoxin (125I-alpha BGT) binding to the reconstituted liposomes was also reduced, although 125I-alpha BGT binding at equilibrium was not affected. Agonist-induced desensitization of the receptor was also affected by these mAbs. mAb 148 binds to the beta subunit of receptor, and mAb 168 binds to the gamma subunit. Both mAbs bind to the cytoplasmic surface of the receptor; correspondingly, both block function when added before reconstitution, and both were found to have no effect on function when added to preformed vesicles. Their effects were not due to interference with the reconstitution process. Both mAbs were capable of cross-linking receptors. In contrast to the bivalent mAbs, monovalent Fab fragments of these two mAbs had little effect on receptor function, which suggests that the effects of the bivalent mAbs resulted primarily from cross-linking receptors.     

Conformation of cytoplasmic segments of acetylcholine receptor alpha- and beta-subunits probed by monoclonal antibodies: sensitivity of the antibody competition approach.

The conformation of the cytoplasmic side of Torpedo marmorata acetylcholine receptor (AChR) was investigated by 22 monoclonal antibodies (mAbs) binding to known sites on the amino acid sequences 339-378 and 336-469 of the AChR alpha- and beta-subunits respectively. Competitions among these mAbs for binding on the intact AChR were compared with their competition for binding on the SDS-denatured subunits and with their corresponding epitopes previously determined on the primary structure of the subunits. We found the following: The three approaches correlated very well suggesting that these mAbs bind on the intact AChR at the same sequences determined by synthetic peptides and not on irrelevant discontinuous epitopes; this finding supports conclusions of Ratnam et al. (1986a) that the amphipathic helix M5 is exposed on the cytoplasmic side of the AChR. The subunit segments alpha 339-378 and beta 336-469 seem to be extended over large distances on the cytoplasmic surface of the AChR. The cytoplasmic surface of beta-subunit has a very immunogenic region. The mAb-competition technique is very sensitive since mAbs to epitopes separated by only about seven residues did not exclude each other, and mAbs to overlapping epitopes exhibited differential competitions with other mAbs.     

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles

In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence beta 429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the beta subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the alpha subunit by a more direct approach. We synthesized peptides including the sequences alpha 330-346, alpha 349-364, alpha 360-378, alpha 379-385, and alpha 389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to alpha subunits between two and six which bound specifically to each of these peptides, except alpha 389-408. We obtained antibodies specific for alpha 389-408 both from antisera against the denatured alpha subunit and from antisera made against the peptide. These antibodies were specific to alpha 389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (alpha 409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic transmembrane domain M5 (alpha 364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathy profile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data.     

Anti-idiotypic antibodies elicited by pterin recognize active site epitopes in dihydrofolate reductases and dihydropteridine reductase

Monoclonal antibodies (mAbs) against antipterin immunoglobulin and dihydropteridine reductase (DHPR) and also polyclonal antibodies against human dihydrofolate reductase (DHFR) were obtained. The anti-idiotypic mAbs and anti-DHPR mAbs bind specifically to human DHFR, Escherichia coli DHFR, soybean seedling DHFR, and human DHPR in solid-phase immunoassays. Further, the mAbs bind to the native but not to the denatured forms of DHFRs. The monoclonal antibodies also inhibit the enzymatic activity of human DHFR but not that of human DHPR. Competitive solid-phase immunoassays show stoichiometric inhibition by methotrexate and partial inhibition by NADPH of mAb binding to human DHFR. Cyanogen bromide fragments derived from human DHFR (residues 15-52 and 53-111), containing several active site residues, bind partially to some of the monoclonal antibodies. Accordingly, polyclonal antibodies to peptide 53-111 of human DHFR cross-react to some extent with human DHPR. Data from competitive immunoassays in which the binding of the various mAbs was tested singly and in combination with other mAbs suggest that these antibodies bind to a common region on human DHFR. The results also indicate that the mAbs display some heterogeneity with respect to specific epitopes. These data suggest that despite the absence of significant amino acid sequence homologies among the various DHFRs and DHPR, they have a fundamentally similar topography at the site of binding of the pterin moiety that is recognized by the anti-idiotypic mAbs generated by pterin. In the relatively simple structure of the pterin ring system there are different substituent groups at positions C4 and C6 in methotrexate, 7,8-dihydrofolate, and 7,8-dihydrobiopterin, suggesting that these antibodies are specific for regions on various proteins that interact with the remainder of the pterin moiety. These mAbs and similar mAbs specified by substituent groups on pterin may thus be used as specific probes or inhibitors of various folate-dependent enzymes and transport proteins. They should also provide insights into some of the general features of antibody recognition of protein antigens.     

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.     

Mapping of phosphorylation-dependent anti-tau monoclonal antibodies in immunoblots using human tau-constructs synthesized by native chemical ligation

In Alzheimer’s disease (AD) neurofibrillary tangles (NFT) are formed by hyperphosphorylated microtubule-associated tau protein. It is still a matter of controversy which phosphorylation sites are AD-specific and how these might be linked to the cause or progress of the disease. Whereas most research projects in this field rely on phosphorylation-dependent tau-specific monoclonal antibodies (mAbs), the phosphorylation patterns recognized by these mAbs are often not characterized in detail. Therefore, we synthesized unphosphorylated, two monophosphorylated (pThr231, pSer235), and the bisphosphorylated (pThr231 + pSer235) tau226-240 peptides. The phosphopeptides were ligated via an N-terminal cysteine to the thioester-activated C-terminus of human aldo/keto reductase AKR1A1. After purification by preparative gel electrophoresis, the ligation products were analyzed by Western blotting and probed with phosphorylation-dependent anti-tau mAbs HPT-101, HPT-103, HPT-104, and HPT-110. The obtained specificities were very similar to the data obtained by ELISA, showing that ELISA-based epitope mapping studies are also valid for immunoblot analyses.     

Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor

Fourteen clonal hybridoma lines that secrete monoclonal antibodies (mabs) to the Torpedo acetylcholine receptor (AChR) have been isolated. When analyzed by an immunoreplica technique, two mabs recognized the alpha subunit, three reacted with the beta subunit, one reacted with the gamma chain, and five recognized the delta subunit. One mab failed to react with any of the subunits using this assay and two mabs recognized determinants found on both the gamma and the delta subunits. These were classified according to their reactivities with the membrane-bound Torpedo AChR. One category is comprised of mabs (including both anti-alpha mabs) that recognize extracellular epitopes. A second classification included mabs that are unable to bind the membrane-associated AChR. The third category is comprised of mabs directed against cytoplasmic epitopes of the AChR. The latter mabs, all of which recognize the gamma or delta subunits or both, bind only slightly to sealed, outside-out Torpedo vesicles. The binding is increased 10-20-fold by either alkaline extraction or treatment of the vesicles with 10 mM lithium diiodosalicylate but not by permeabilization of the vesicles with saponin. Three of the six mabs in this category react with frog muscle endplates but only if the cytoplasmic surface of the membrane is accessible.     

Construction and characterization of single-chain antibodies against human insulin-like growth factor-I receptor from hybridomas producing 1H7 or 3B7 monoclonal antibody

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.     

Antipeptide antibodies reveal interrelationships of MBP 200 and MBP 235: unique apoB-specific receptors for triglyceride-rich lipoproteins on human monocyte-macrophages

Two human monocyte-macrophage (HMM) membrane binding proteins, (MBP) 200 and 235, are receptor candidates that bind to the apolipoprotein (apo)B-48 domain in triglyceride-rich lipoproteins for uptake independent of apoE. Microsequence analysis of the purified reduced MBP 200R characterized tryptic peptides of MBP 200R. A synthetic peptide mimicking a unique, unambiguous 10-residue sequence (AEGLMVTGGR) induced antipeptide antibodies that specifically recognized MBP 200, 235 and 200R, in 1- and 2-dimensional analyses, indicating 1) the ligand binding protein was sequenced and 2) MBP 200 and 235 yielded MBP 200R upon reduction. These antibodies identified the MBPs in human blood-borne, THP-1, U937 MMs, and endothelial cells (EC) but not in human fibroblasts or Chinese hamster ovary (CHO) cells. Fluorescence activated cell sorting (FACS) analysis located the MBPs on the MM surface as necessary for receptor function. The 10-residue, unambiguous MBP 200-derived sequence is unique, with no matches in extant protein databases. Antipeptide antibodies bind to the MBPs in reticuloendothelial cells that have this receptor activity, but not to proteins in cells that lack this receptor activity. These studies provide the first direct protein sequence and immunochemical data that a new, unique apoB receptor for triglyceride-rich lipoproteins exists in human monocytes, macrophages, and endothelial cells.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration that lysine-501 of the alpha polypeptide of native sodium and potassium ion activated adenosinetriphosphatase is located on its cytoplasmic surface

Evidence that the peptide HLLVMKGAPER, which can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion, is located on the cytoplasmic surface of the native enzyme has been obtained. An immunoadsorbent directed against the carboxy-terminal sequence of this tryptic peptide has been constructed. The peptide KGAPER was synthesized by solid-phase techniques. Antibodies against the sequence -GAPER were purified by immunoadsorption, using the synthetic peptide attached to agarose beads. These antibodies, in turn, were coupled to agarose beads to produce an immunoadsorbent. Sealed, right-side-out vesicles, prepared from canine kidneys, were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin, respectively. A tryptic digest of these labeled vesicles was passed over the immunoadsorbent. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digests obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction as applied to these vesicles, it could be concluded that this increase in incorporation resulted only from the access that the reagents gained to the inside of the vesicles in the presence of saponin and that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.     

Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles.

Synaptophysin is a major integral membrane protein of synaptic vesicles. Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy-terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure. The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin. By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure. Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.     

Monoclonal antibodies against defined epitopes of the human transferrin receptor cytoplasmic tail.

Three murine monoclonal antibodies (mAbs) against the 61-residue amino-terminal cytoplasmic tail of the human transferrin receptor (TR) have been produced by immunization of mice with recombinant human TR produced in a baculovirus expression system. Mutant human TRs expressed in chick embryo fibroblasts (CEFs) with point mutations or deletions in their cytoplasmic tails have been used to map the epitopes defined by each of the mAbs. One mAb, H68.4, previously shown to block receptor internalization, binds proximal to the carboxy-terminal side of the YTRF internalization signal of TR. The second mAb, H73.2, binds near to the carboxy-terminal side of the H68.4 epitope, whereas the third mAb, 160.1, binds closer to the transmembrane region. H68.4 and H73.2 are auto-antibodies consistent with their epitopes mapping to a region of the human TR that has an identical amino acid sequence to the mouse TR. All three mAbs crossreact with the cytoplasmic tail of Chinese hamster TR. Double labelling of recombinant human TRs on chick embryo fibroblast (CEF) cell membrane preparations with B3/35 and H68.4 antibody-gold conjugates established that receptors in clathrin-coated pits were not labeled with H68.4, implying that associated coated pit proteins may block binding of this mAb.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

TGN38/41: a molecule on the move

TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN.