location of ligand-binding sites on the nicotinic ... · location of ligand-binding sites on the...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 hy The American Society of Biological Chemists, h e .

Vol. 261, No. 29, Issue of October 15, pp. 13735-13743,1986 Printed in U. S. A.

Location of Ligand-binding Sites on the Nicotinic Acetylcholine Receptor &-Subunit*

(Received for publication, February 21, 1986)

Steen E. Pedersen, Evan B. DreyerS, and Jonathan B. Cohenj From the Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 631 10

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) a-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR a-subunit by Staphy- lococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-a-benzyltrimethylammonium iodide-reactive site and binds l2‘1-

a-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the a- subunit.

Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-8-N-acetylglucosaminidase H, consistent with the presence of N- linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the a-subunit sequence.

Experiments designed to map disulfide bonds within the AChR a-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys- 192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.

The nicotinic acetylcholine receptor (AChR’) isolated from

*This research was supported in part by United States Public Health Service Grant NS-19522, a grant from the Muscular Dystro- phy Association (to J.B.C.), and National Institute of General Med- ical Sciences Postdoctoral Fellowship GM-10093 (to S.E.P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Predoctoral Training Grant GM-07306 (to Harvard Medical School). Present address: Dept. of Ophthalmology, Massa- chusetts Eye and Ear Infirmary, Boston, MA 02114.

To whom correspondence should be addressed Dept. of Anatomy & Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. ’ The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; MBTA, 4-(N-maleimido)-a-benzyltrimethylammonium iodide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FITC-ConA, fluorescein isothiocyanate- labeled concanavalin A, Endo H, endo-8-N-acetylglucosaminidase H; V8 protease, Staphylococcus aureus V8 protease; BSA, bovine serum albumin.

Torpedo electric organ contains a cation-selective ion channel whose opening is regulated by the binding of acetylcholine (ACh) (for review, see Popot and Changeux, 1984; McCarthy et al., 1986). Analysis of the mechanism of permeability control by the AChR requires the localization of the agonist- binding site within the known amino acid sequence of the AChR (Noda et al., 1982,1983a, 198313) as well as the identi- fication of the structure of the ion channel itself. In addition to the site of binding of ACh (and competitive antagonists such as tubocurare), the Torpedo AChR contains a distinct allosteric antagonist site that binds aromatic amine noncom- petitive antagonists (Cohen et al., 1985). Although it is not known whether the allosteric antagonist site is a part of the ion channel itself or a distinct regulatory site, electrophysio- logical studies of the actions of these compounds (Peper et al., 1982; Neher, 1983) yield results consistent with the notion that they bind to a site within the structure of the ion channel.

Ligand-binding sites have been localized within the AChR by the development of specific affinity labels. The ACh- binding sites have been localized to the a-subunits by the compounds 4 - (N-maleimido) - a -benzyltrimethylammonium iodide (MBTA) and bromoacetylcholine, which bind to the ACh-binding site and react with a sulfhydryl in the vicinity of the binding site, generated by reduction of a labile disulfide bond within the a-subunit. These compounds label preferentially one of the a-subunits (Damle and Karlin, 1978; Wolosin et al., 1980), and MBTA labels preferentially the high affinity d-tubocurare-binding site (Neubig and Cohen, 1979; Dreyer, 1984).

Affinity labels have also been used in attempts to identify the location of the allosteric antagonist-binding site. However, the labeling pattern is dependent on the compound used, and specific labeling of each subunit has been demonstrated. Tri- methisoquin, trimethisoquin azide, perhydrohistrionicotoxin, and phencyclidine can be specifically incorporated into the 6- subunit of T . marrnorata AChR by UV irradiation (Oswald and Changeux, 1981a, 1981b). In contrast, chlorpromazine will react specifically with all the subunits (Heidmann et al., 1983). Two analogues of quinacrine, quinacrine mustard (Kal- dany and Karlin, 1983) and quinacrine azide (Cox et al., 1985), react specifically with the a- and P-subunits. Meproadifen mustard, characterized in the preceding paper (Dreyer et aL, 1986), also reacts specifically with the a- and @-subunits.

Proteolytic mapping (Cleveland et al., 1977) provides an initial approach to identify regions of the AChR subunits that contribute to ligand-binding sites. To identify regions of the a-subunit alkylated by the allosteric antagonist [3H]meproadifen mustard, we report here analysis of the digestion pattern produced by Staphylococcus aureus V8 protease (V8 protease). Interpretation of the map is facilitated by comparison to the pattern generated by [3H]MBTA that reacts with CysIs2- Cyslg3 of the a-subunit (Kao et al., 1984) and to the location of N-linked carbohydrate. In addition, to extend the map of

13735

13736 Binding Sites of the Acetylcholine Receptor

the ACh-binding site without use of sulfhydryl-specific affinity reagents, [3H]tubocurare is used as a photoaffinity reagent.

Gullick et ul. (1981) reported that digestion of the AChR a- subunit with V8 protease yielded two predominant cleavage fragments with apparent molecular masses of 20 and 18 kDa; [3H]MBTA was associated with the 20-kDa peptide, carbohydrate with the 18-kDa peptide, and certain monoclonals bound to both peptides. Because these two large fragments appeared identical with respect to N-terminal primary sequence, both beginning at Val-46 (Conti-Tronconi et al., 1984), it was proposed that the two a-subunits of the AChR differ with respect to carbohydrate content and reactivity to [3H]MBTA. However, Wilson et ul. (1984,1985) reported that V8 protease generated a 20-kDa peptide binding MBTA and ‘2sI-a-bungarotoxin that had an N terminus beginning after Asn-141 of the a-subunit sequence. In view of these discrep- ancies, to analyze the labeling pattern of [3H]meproadifen mustard or [3H]tubocurare after V8 protease digestion of AChR a-subunit, individual fragments were characterized by N-terminal amino acid analysis, by [3H]MBTA reactivity, by the ability to bind fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA), and by their sensitivity to endo- P-N-acetylglucosaminidase H (Endo H). [3H]Meproadifen mustard and [3H]tubocurare as well as [3H]MBTA are found to react with sites on the 20-kDa peptide that is shown by N- terminal sequence analysis to begin a t Ser-173 of the a- subunit sequence. N-linked carbohydrate is found associated with the 18-kDa peptide that begins at Val-46 of the a-subunit sequence. These results are discussed with reference to possible models of ligand-binding sites within the a-subunit. Results with implications for possible disulfide linkages within the a-subunit are also presented.

EXPERIMENTAL PROCEDURES~

RESULTS

Effect of Endo H on AChR-Endo H releases high mannose and hybrid-type Asn-linked oligosaccharides from glycopro- teins (Varki and Kornfeld, 1983), and treatment of SDS- solubilized AChR-rich membranes with Endo H increases the mobility of all the subunits of the AChR when analyzed by SDS-PAGE (Fig. 1). The a-, p-, and y-subunits were quantitatively converted from lower to higher mobility entities by Endo H, whereas the &subunit was apparently only partially converted. Other proteins present in the membrane suspension were insensitive to Endo H, notably the 43-kDa protein, as well as polypeptides of 97, 37, 31, and 29 kDa that were present in contaminating membrane fractions (Jeng et ul., 1981).

If the AChR was labeled with [3H]MBTA, [3H]meproadifen mustard, or [3H]meproadifen mustard in the presence of excess meproadifen (to control for nonspecific labeling by [3H]meproadifen mustard) prior to treatment with Endo H, there was no effect on the changes in mobility induced by Endo H. The fluorogram in Fig. 1B shows membranes labeled with [3H]meproadifen mustard (lanes 3-6) or [3H]MBTA (lunes 7 and 8). [3H]MBTA labeled only the a-subunit, and the [3H]MBTA-labeled subunit was quantitatively converted to the high mobility form by Endo H. Although [3H]meproad-

The “Experimental Procedures” are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-572, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1 2 3 4 s

6- Y- P-

43 K- a- .

-116 -97

-66

-45

-29

FIG. 1. Effect of Endo H on membranes labeled with [3H] meproadifen mustard or [3H]MBTA. AChR-rich membranes were not labeled (lanes 1 and 21, labeled with 15 pM [3H]meproadifen mustard in the absence (lanes 3 and 4 ) or presence (lanes 5 and 6 ) of 200 p~ meproadifen, or labeled with [3H]MBTA (lanes 7 and 8) as described under “Experimental Procedures.” Samples were incubated without Endo H (lanes 1,3, 5, and 7) or with 1.5 milliunits of Endo H (lanes 2, 4, 6, and 8). Aliquots representing 25 pg of membranes were electrophoresed on an 8% SDS polyacrylamide gel. The gel was stained with Coomassie Blue (panel A ) and then processed for fluorography (panel B, 16-h exposure). Migration of molecular mass standards is indicated on the right for P-galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). AChR subunits and the non-receptor 43-kDa protein are identified on the left.

ifen mustard alkylates several polypeptides in addition to AChR subunits, only alkylation of the a- and P-subunits is specific, i.e. inhibited by the presence of 0.2 mM meproadifen (compare lanes 3 and 4 to lanes 5 and 6). As for [3H]MBTA, AChR a- and p- subunits alkylated by [3H]meproadifen mustard were converted quantitatively to the high mobility form by Endo H.

The V8 Cleavage Puttern-To map the ligand-binding sites on the a-subunit, we used the procedure of Cleveland et al. (1977) to analyze the peptide pattern generated with S. uureus V8 protease, which cleaves specifically at the carboxy side of glutamate residues. In Fig. 2 are shown the cleavage patterns generated from a-subunits isolated by slab gel electrophoresis with respect to Coomassie Blue stain (Fig. 2 A ) and incorporated radiolabel as detected by fluorography (Fig. 2B) after no labeling (lunes 1 and 2), labeling with [3H]MBTA (lunes 3 and 4, labeling with [3H]meproadifen mustard (lanes 5 and 6), or labeling with [3H]meproadifen mustard in the presence of 0.2 mM meproadifen (lunes 7 and 8). Each sample was treated without (odd lanes) or with (even lanes) Endo H. Coomassie Blue stain of the gel (Fig. 2 A ) reveals that, in the absence of Endo H treatment, there were two predominant stable cleavage products generated with apparent molecular masses of 20 kDa (V8-20) and 18 kDa (V8-18). There were also minor cleavage products at about 10 kDa (V8-10) and additional poorly resolved material near the bottom of the gel. In the experiment presented in Fig. 2, V8 protease migrates as a single major band with apparent molecular mass of 27 kDa with a minor band at 8 kDa.

When the a-subunit was treated with Endo H prior to cleavage with V8 protease, the cleavage pattern was altered (lane 2). The upper band, V8-20, was apparently unaffected, but there was no band at the position normally occupied by V8-18 and two new bands with apparent molecular masses of

Binding Sites of the Acetylcholine Receptor 13737

1 2 3 4 5 6 7 8

P-

2 1. 20- - 9 4

18- w 1

15- 12- 0

10-

1 2 3 4 5 6 7 8 ..-c ._.I I. -

-45

-29

-21

-1 8 -1 2

FIG. 2. Digestion pattern of AChR a-subunit by Vt3 protease. Membranes were labeled with [3H]meproadifen mustard or [3H]MBTA and then treated with Endo H (4.5 milliunits) as described under “Experimental Procedures.” Polypeptides resolved by slab gel electrophoresis were visualized by brief staining with Coomassie Blue. The bands corresponding to AChR a-subunit were excised and placed in the mapping gel according to procedure A under “Experimental Procedures.” Each gel piece was overlaid with 3 pg of V8 protease. The gel pattern revealed by Coomassie Blue staining is shown in panel A and the fluorogram in panel B (5-day exposure). AChR a- subunit was unlabeled (lanes 1 and 2), or labeled with [3H]MBTA (lanes 3 and 4) , with [3H]meproadifen mustard (lanes 5 and 6) , or with [3H]meproadifen mustard in the presence of 0.2 mM meproadifen (lanes 7 and 8). Odd-numbered lanes contain samples not treated with Endo H whereas even-numbered lanes contain samples treated with Endo H. Migration of molecular mass standards is indicated on the right. The standards are ovalbumin (45 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (21 kDa), myoglobin (17.7 kDa), and cytochrome c (12 kDa). The nomenclature used for the various species observed on the gel is indicated on the left. P indicates V8 protease.

15 (V8-15) and 12 (V8-12) kDa were generated. The simplest interpretation of this result was that V8-18 was derived from part of the a-subunit that contained carbohydrate and that V8-12 (the more intense of the novel bands) represented the deglycosylated form of V8-18. This does not readily explain the presence of V8-15 (but see the sections below). The result indicates that V8-18 contains the N-linked glycosylation site on the a-subunit.

Examination of the fluorogram revealed that Endo H treatment had no effect on the location of the [3H]MBTA label within the gel (lanes 3 and 4, Fig. 2B) and that the label was associated with V8-20. This result established that an N- linked carbohydrate and the [3H]MBTA-labeling site were contained within different a-subunit fragments generated by V8 protease.

The labeling pattern generated by [3H]meproadifen mustard also was not affected by Endo H (compare lanes 5 and 6, Fig. 2B); however, the radioactivity was not associated with either V8-18 or V8-20, but rather with a band migrating with an apparent molecular mass of 21 kDa (V8-21). That the band appearing in the autoradiogram from [3H]meproadifen mustard did not co-migrate with V8-20 is clear from comparison of the [3H]MBTA and the [3H]meproadifen mustard labeled tracks (lanes 3-6, Fig. 2B). Although V8-21 was only faintly visible by Coomassie Blue staining, it was present in the gel only after labeling with meproadifen mustard and not when alkylation had been blocked with 0.2 mM meproadifen (Fig.

2, lunes 7 and 8), (see also Fig. 3). V8-21 was more obvious after silver-staining a gel from a similar experiment (data not shown).

The limited formation of V8-21 (as judged by Coomassie Blue stain) would be expected if [3H]meproadifen mustard alkylated only a small fraction of the available sites. Under the condition described in Fig. 2, about 7% of the a-subunits were actually labeled. Either the specific labeling of AChR a- subunit by [3H]meproadifen mustard causes a change in the mobility of the fragment that is labeled, or the presence of [3H]meproadifen mustard attached covalently to the a-subunit alters the sites of cleavage by V8 protease, yielding a new cleavage pattern. Since V8-21 was Endo H-insensitive, we suspected that V8-21 was similar to V8-20, and therefore would also contain the MBTA-reactive site. This hypothesis was tested using a double-label protocol.

Torpedo AChR membranes were labeled with nonradioactive meproadifen mustard and then with [3H]MBTA. AChR a-subunits were isolated and subjected to proteolysis by V8 protease. The resultant gel was visualized by Coomassie Blue stain (Fig. 3A) and by fluorography (Fig. 3B). When membranes were labeled with nonradioactive meproadifen mustard alone (lane I ) , V8-21 can be seen in the Coomassie Blue stain, but not when the alkylation reaction was conducted in the presence of 0.2 mM meproadifen (lune 2). When membranes were labeled with meproadifen mustard and [3H]MBTA (lunes 3 and 4 ) , there was no change in the Coomassie Blue staining pattern from lunes 1 and 2, but the fluorogram shows that radioactivity appears in bands corresponding to V8-21 and

1 2 3 4 5 1 2 3 4 5 -2 9

P-

21- 20- 18-

p7 10-

-21

-18 -1 2

FIG. 3. Double labeling of a-subunit with [3H]MBTA and nonradioactive meproadifen mustard. AChR-rich membranes (200 pg) were labeled with 20 PM nonradioactive meproadifen mustard as described under “Experimental Procedures” (Step 1). The membranes were washed twice by centrifugation and resuspension and then trace-labeled with 0.75 pCi (25 pmol) of [3H]MBTA (Step 2). The a-subunits were isolated by slab gel electrophoresis and placed in the proteolytic cleavage gel map according to procedure A under “Experimental Procedures.” Panel A displays the Coomassie Blue staining pattern and panel B displays the fluorogram (1-day exposure). Lanes 1 and 2 contain membranes labeled as above but with omission of [3H]MBTA from Step 2. Lane 2 contains membranes whose meproadifen mustard-labeling was blocked by inclusion of 200 p~ meproadifen in Step 1. Lanes 3 and 4 are similar to lanes I and 2 except that [3H]MBTA was included in Step 2. Lane 5 contains a control sample where meproadifen mustard was excluded from Step 1. Migration of the molecular mass standards is indicated on the right. Top and bottom portions of the gel containing no material are not shown.


V8-20 (lane 3) and that no radioactivity appears in the bands corresponding to V8-21 when 0.2 mM meproadifen was present during the labeling by mustard (lane 4) . The Coomassie Blue stain and fluorogram observed in the presence of excess meproadifen (lane 4 ) was the same as that observed where membranes were labeled with [3H]MBTA alone (lane 5) .

Localization of Carbohydrate with Concanavalin A-A1- though Endo H treatment of AChR a-subunit prior to cleavage with V8 protease strongly indicated that the Endo H- sensitive carbohydrate was associated with fragment V8-18, it was possible to directly localize carbohydrate using FITC- ConA. ConA binds mannose and glucose residues. Using the gel overlay method of Burridge (1978), gels were incubated with FITC-ConA and the fluorescent bands were visualized over UV light. For these experiments, purified [3H]MBTA- labeled a-subunit was used, rather than excising a-subunit from individual gel lanes. The a-subunit was mixed directly with V8 protease and sample buffer, and the complete solution was loaded onto the gel as described under “Experimental Procedures.”

In Fig. 4 are shown resultant gel patterns in terms of Coomassie Blue stain (Fig. 4A), fluorescence of bound FITC- ConA (Fig. 4B), and a fluorogram of [3H]MBTA (Fig. 4C). V8-20 and V8-18 appeared to bind FITC-ConA in approxi- mately equal amounts (see lane 3, Fig. 4B). Treatment with Endo H prior to cleavage with V8 protease eliminated FITC- ConA binding to both uncleaved a-subunit (lane 2) and to the cleavage products (lane 4 ) , suggesting that Endo H does remove at least one carbohydrate moiety from the a-subunit and that this moiety binds ConA. FITC-ConA binding to a- subunit or to V8 protease cleavage products could be blocked by incubation in the presence of 0.1 M a-methylmannoside (data not shown), indicating sugar-specific binding of FITC- ConA.

Several results suggested that the carbohydrate-binding

1 2 3

a

P-

20- ~- 18- 1 5- 12-

10-

A 4 5 6

entity migrating at V8-20 was not the [3H]MBTA-labeled peptide but an incompletely cleaved form of V8-18 that co- migrated with V8-20. Endo H treatment altered the mobility of V8-18 and destroyed FITC-ConA binding, but did not alter the mobility of the [3H]MBTA-labeledpeptide (Fig. 4C). Endo H treatment yielded two new peptides in the cleavage pattern (V8-15, V8-12) (see lanes 4 and 6, Fig. 44). These two new peptides had nearly the same difference in mobility that V8- 18 and V8-20 did. Therefore, it was plausible that V8-15 corresponded to the deglycosylated form of a peptide that co- migrated with the [“IMBTA-labeled peptide at V8-20 and that V8-12 was the deglycosylated form of V8-18.

The amount of FITC-ConA binding to the position at V8- 20 was strongly dependent on the buffer composition of the sample loaded onto the cleavage gel. Lanes 5 and 6 of Fig. 4, A, B, and C, show the cleavage pattern when the sample was loaded in overlay buffer rather than in Laemmli sample buffer. FITC-ConA bound only to V8-18 (lane 5, Fig. 4B). Accord- ingly, after treatment with Endo H, V8-12 was more promi- nent than V8-15 (compare lane 6 to lane 4 in Fig. 4A). Our conclusion that the binding of FITC-ConA to V8-20 represented binding to an incompletely cleaved form of V8-18 and that the extent of complete cleavage to the 18-kDa species was dependent on the sample buffer was corroborated by N- terminal amino acid sequencing of the relevant peptides, as is described below.

N-terminal Amino Acid Sequencing of V8-18, V8-20, and V8-15-To determine where in the primary amino acid sequence each of the peptides fit, V8-20, V8-18, and V8-15 were isolated and submitted to N-terminal Edman degradation using an Applied Biosystems gas phase sequencer.

Peptides from each band were isolated as described under “Experimental Procedures.” Peptides from band V8-20 were isolated from a-subunit that had been treated (or not) with Endo H. V8-18 was isolated from a-subunit with carbohydrate

C 1 2 3 4 5 6

-45

-29

.2 1

.18

.12

FIG. 4. Binding of FITC-ConA by AChR a-subunit and its proteolysis fragments. Purified a-subunit, isolated by preparative gel electrophoresis, was concentrated to 1 mg/ml and then incubated in the absence or presence of 3 milliunits of Endo H as described under “Experimental Procedures.” Proteolysis was carried out by mixing the a-subunit (10 pg) with V8 protease (2 pg) and dilution (5-fold) into one of two buffers immediately prior to being loaded onto the gel: Buffer 1 (lanes 1-4) consisted of Laemmli sample buffer containing 2.5% 2- mercaptoethanol (essentially procedure B under “Experimental Procedures”); buffer 2 (lanes 5 and 6 ) consisted of overlay buffer without reducing agent. Lanes 1,3, and 5 contained a-subunit not digested with Endo H, and lanes 2 ,4 , and 6 contained Endo H-digested a-subunit. Lanes 1 and 2 did not include V8 protease; lanes 3-6 included 2 pg of V8 protease. The gap between lanes 4 and 5 represents wells that were not loaded to avoid diffusion of buffer 1 into buffer 2, or conversely. The gel was incubated with FITC-ConA, as described under “Experimental Procedures.” The fluorescent pattern is displayed in panel B. The gel was then stained with Coomassie Blue (panel A ) and processed for fluorography (panel C ) (1-day exposure). The relevant bands are identified on the left. Migrations of molecular mass standards are shown on the right.


still attached and V8-15 from Endo H treated a-subunit. After precipitation with cold acetone, the peptides were assayed for protein and analyzed for homogeneity by SDS-PAGE. Typical protein recoveries were 10-20 pg, representing about 1 nmol of peptide compared to the 4 nmol of a-subunit loaded onto the gel. The losses were due in part to incomplete cleavage by V8 protease, because significant quantities of incompletely cleaved material were seen in the gel. Some loss also resulted as bands were excised from gels, because we wished to avoid contamination by other peptides. The isolated peptides ran true on SDS-PAGE, except that some oligomerization and breakdown of V8-20 was observed upon prolonged storage (data not shown).

Table I displays the results of the first six sequencing cycles for each of the bands examined and the yield (in picomoles) of each amino acid obtained from the sequenator. Also displayed are the expected sequences for peptides having N termini at Val-46, Ser-162, and Ser-173 of the AChR a- subunit. When a-subunit that had not been digested with Endo H was used, two sequences were observed from V8-20, corresponding to the N termini at Val-46 and Ser-173. When a-subunit that had been treated with Endo H was used to prepare V8-20, the sequence beginning at Val-46 did not appear; the major sequence began at Ser-173, and, in addition, a minor sequence starting at Ser-162 was seen. The sequences observed for V8-18 and V8-15 were identical, both beginning at Val-46. The appearance of the V8-18 sequence (Val-46 et seq.) in V8-20, when V8-20 was derived from a-subunit that had not been treated with Endo H, was consistent with the expectation from the FITC-ConA binding experiment-that an incompletely cleaved form of V8-18 co-migrates with V8- 20 and could be removed with Endo H treatment of the a- subunit prior to V8 cleavage.

Determination of the d-Tubocurare-binding Site-The competitive antagonist [3H]d-tubocurare can be incorporated into membranes when irradiated with UV light (Dreyer, 1984). UV irradiation results in incorporation of label into all the AChR subunits and some contaminating proteins (Fig. 5, lanes 1 and 3) , but only the incorporation into the a-subunit was specific, as shown by the inhibition of incorporation by 1 mM carbamylcholine (lanes 2 and 4 ) . The amount of incorporation reflects labeling of about 0.1% of the a-subunits. Membranes from both T. califoornica (lanes 1 and 2) and T. marmorata (lanes 3 and 4 ) can be specifically labeled with [3H]d-tubocurare.

Cycle

The V8 protease cleavage pattern of [3H]d-tubocurare- labeled a-subunit is displayed in Fig. 6. The Coomassie Blue- stained pattern was unaltered by labeling with [3H]d-tubocurare (Fig. 6A). Examination of the fluorogram of the digestion pattern (Fig. 6B) clearly indicated that the specifically incorporated label was associated with V8-20 (compare lanes 1 and 2 to 3 and 4 ) , and that treatment of a-subunit with Endo H (compare lunes 1 and 2) did not affect the mobility of the label after cleavage with V8 protease. Thus, V8-20 also contributes to the d-tubocurare-binding site.

Determination of the a-Bungarotoxin Site-a-Bungaro- toxin binds to isolated a-subunit and fragments of the a- subunit (Gershoni et al., 1983; Wilson et al., 1984). We utilized the procedure developed by Gershoni et al. (1983) to demonstrate that '251-oc-bungarotoxin binds to the a-subunit fragment designated V8-20. Prior treatment of the a-subunit with Endo H had no effect on the binding of '251-a-bungarotoxin to the proteolytic fragment, substantiating that the binding was to V8-20 and not to V8-18. '251-a-bungarotoxin binding could be blocked by d-tubocurare or by excess unlabeled a- bungarotoxin (data not shown).

Disulfide Bonding within the a-Subunit-The a-subunit contains seven cysteines, none of which are involved in inter- subunit disulfide bonds (reviewed in McCarthy et al., 1986). Since the sulfhydryls of the intact AChR a-subunit react with iodoacetamide or N-ethylmaleimide only after exposure to disulfide-reducing agents (de Souza Otera and Hamilton, 1984), disulfide bonds exist within each a-subunit. One of the disulfides is in the vicinity of the AChR-binding site itself, since [3H]MBTA alkylates Cys-192 or Cys-193 only after reduction (Kao et al., 1984). Several models, based on the primary amino acid sequence, have suggested that Cys-192 and Cys-193 are disulfide-linked to Cys-128 and Cys-142 (Boulter et al., 1985; Kao et al. 1984; Criado et al., 1985). An alternative model suggests that Cys-142 and Cys-128 form a disulfide bond (Finer-Moore and Stroud, 1984) and that Cys- 192 and Cys-193 may form another disulfide bond. There exists some evidence consistent with the latter possibility (Kao and Karlin, 1986).

Analysis of the V8 protease cleavage pattern of the a- subunit suggested that these two models should be distin- guishable. V8-20 contained Cys-192, Cys-193, and Cys-222, since these residues are near the N terminus of this fragment. V8-18 contained Cys-128 and Cys-142 because these residues lie beyond the N terminus at Val-46 and before the first V8

TABLE I N-terminal amino acid sequence analysis of AChR a-subunit proteolytic peptides

Peptides were prepared for sequencing as described under "Experimental Procedures." The isolated [3H]MTBA- labeled a-subunit (200 pg) was either not treated, or treated with Endo H (6 milliunits) prior to electrophoresis of the proteolytic gel. Peptides isolated from the appropriate bands ran true upon re-electrophoresis on a 15% SDS polyacrylamide gel. The quantities of peptide submitted for sequence analysis were assessed by measuring protein and measuring [3H]MBTA that had been incorporated (for V8-20 only). The amounts sequenced were: (- Endo H) V8-18,15 pg; V8-20,9 pg; (+ Endo H) V8-15, l l p g ; V8-20,17 pg.

- Endo H Expected sequences + Endo H for peptides starting

at amino acid

V8-18 V8-20 V8-15 V8-20

Residue umol Residue umol Residue omol Residue omol 46 1 73 162

1 Val 57 Ser/Val NIa/190 Val 300 Ser 200 Val Ser Ser 2 Asn 54 Gly/Asn 97/118 Asn 280 Gly/Asp 370/80 Asn Gly Asp 3 Gln 31 Glu/Gln 126/100 Gln 290 Glu 280 Gln Glu Arg 4 Ile NI" Trp/Ile NI/75 Ile 200 Trp/Pro 100/125 Ile Trp Pro 5 Val 45 Val 188 * Val 235 Val/Asp 250/80 Val Val Asp

. ~~~-~

6 Glu NI Met/Glu 74/108 Glu 136 Met/Leu 290/112 Glu Met Leu NI, peak was not integrated.


1 2 3 4

6 Y P

43 a

FIG. 5. Photoaffinity labeling of AChR-rich membranes with [SH]d-tubocurare. AChR-rich membranes from T. californica (lanes 1 and 2) or T. marmorata (lanes 3 and 4 ) were photoaffinity labeled with 12 p~ [3H]d-tubocurare in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 mM carbamylcholine as described under “Experimental Procedures.” A fluorogram exposed for 4 days is shown.

protease cleavage site beyond the cardohydrate attachment site at Asn-141. Thus, if the first model is correct, then a V8 cleavage carried out in the absence of reducing agent should result in a peptide of lower mobility that contained both V8- 20 and V8-18. If the second model is correct, then no change in the pattern would be observed in the absence of reducing agent.

The experiment, as shown in Fig. 7, was carried out by electrophoresing two sets of two gels in parallel. The first set (Fig. 7A) had no reducing agent introduced at any point in the procedure, and the second set (Fig. 7B) was executed with sulfhydryl-reducing agent present at all stages of the protocol. Each set of gels consisted of an 8% gel for separation of the AChR subunits followed by a proteolytic cleavage gel.

No difference was observed in the AChR a-subunit digestion products generated by V8 protease from a-subunit isolated and maintained in the absence (lane 1, Fig. 7A) or presence (lane 1, Fig. 7B) of reducing agent. AChR a-subunit that had been electrophoresed on the first gel under nonreducing conditions was also transferred to the second gel and electrophoresed under reducing conditions ( l a n e 2, Fig. 7B). The converse experiment was also performed where a-subunit was isolated in the presence of reducing agent and then transferred to the nonreducing proteolytic cleavage gel (lane

A 1 2 3

P-

20- 18- 15- 12- P- 10-

B 4 1 2 3 4

-29

-21

-18

-1 2

FIG. 6. Localization of the ['Hid-tubocurare-reactive site in the a-subunit. Membranes (about 6 PM ACh-binding sites) were photoaffinity labeled by [3H]d-tubocurare (4 p ~ ) as described under “Experimental Procedures.” Labeling was performed in the absence (lanes 1 and 2) or presence (lanes 3 and 4 ) of 1 mM carbamylcholine. After labeling, the membranes were collected by centrifugation in the airfuge (27 p.s.i.). Membranes were dissolved in 1% SDS and then incubated overnight in the absence (lanes 1 and 3) or presence (lanes 2 and 4 ) of 4.5 milliunits of Endo H. The a-subunits were isolated and submitted to proteolytic mapping as described under ”Experi- mental Procedures,” using procedure A. The gel was stained with Coomassie Blue (panel A ) and processed for fluorography. A 9-day exposure of the fluorogram is shown in panel B. Top and bottom portions of the gel are not shown.

2, Fig. 7A). These patterns were identical to those in lane 1 (Fig. 7). As a control, IgG was included (lanes 3-5 of Fig. 7)) since reduction of native IgG results in the predictable appearance of light and heavy chain within the gel pattern. In no case was any spurious or unexpected reduction observed. The lack of noticeable change of cleavage pattern from a gel electrophoresed under reducing conditions to one electrophoresed under nonreducing conditions indicates that there were no disulfide linkages between any cysteine residues in V8-18 to any in V8-20.

DISCUSSION

The studies presented here provide a characterization of the regions of the AChR a-subunit that contribute to two distinct functional domains of the AChR, the site of binding of ACh (and competitive antagonists) and the allosteric antagonist-binding site. S. aureus V8 protease produced a de- ceptively simple fragmentation of the a-subunit consisting of two predominant peptides of apparent molecular masses of 18 and 20 kDa. The two novel affinity labels examined, [3H] d-tubocurare for the ACh-binding site and [3H]meproadifen mustard for the allosteric antagonist-binding site, as well as [3H]MBTA, each had their reactive site within the 20-kDa peptide, V8-20, beginning at a-subunit Ser-173. The presence of the [3H]MBTA-reactive site within this fragment is consistent with the data of Kao et al. (1984, 1986)) who determined that [3H]MBTA reacts with Cys-192 and Cys-193. In contrast to [3H]MBTA, photoincorporation of [3H]d-tubocurare into a-subunit occurs in the absence of disulfide-reducing reagents, so it is likely that [3H]d-tubocurare alkylates residues in the ACh-binding site distinct from the cysteines labeled by [3H]MBTA. We also demonstrated (in agreement with Neumann et al., 1985 and Wilson et al., 1985) that IZ5I-


A B 1 2 3 4 5 6 1 2 3 4 5 6

H L-

H-

PC

P- L-

-4 5

- 2 9

20- 18-

P- 10-

-21

-18 - 12

V8-P + + - - + b + + - - + b SH-1 - + - + - - + - + - + +

+ + + + + + SH-2 - - - - - - FIG. 7. Comparison of the proteolytic map of the AChR P-

subunit using V8 protease under disulfide-reducing and nonreducing conditions. AChR membranes were subjected to SDS- PAGE in the absence (system A) or presence (system B) of disulfide reducing agents. In system A, Laemmli sample buffer did not contain 2-mercaptoethanol, and sodium thioglycolate was omitted from the electrophoresis buffer. In system B, 2.5% 2-mercaptoethanol was included in the Laemmli sample buffer, and the electrode buffer included 1 mM sodium thioglycolate. Two 250-pg samples of membranes and four samples of 40 pg of IgG were subjected to SDS-PAGE in each of these systems, using 8% acrylamide resolving gels. Bands corresponding to the a-subunit of the AChR (in both systems A and B), the unreduced IgG (in system A), and a heavy chain dimer of IgG (in system B) were excised after brief staining with Coomassie Blue and soaked for >1 h in overlay buffer. If the band was to be electrophoresed in system B (containing reducing agent) for the second electrophoresis step, the overlay buffer also contained 1 mM dithiothreitol. The gel slices were placed into the wells of two identical proteolytic mapping gels. Gel A was electrophoresed in the absence of reducing agent, but the electrophoresis buffer of Gel B contained 1 mM sodium thioglycolate. The gel slices placed in lanes 2 and 4, containing a-subunit and IgG, respectively, were exchanged between systems after excision from the first gel. That is, the gel slices from system A (lanes 2 and 4 ) containing a-subunit or IgG were soaked in overlay buffer with 1 mM dithiothreitol and then electrophoresed in Gel B, under reducing conditions. The converse treatment was applied to gel slices initially run in system B, then placed in system A for the second gel. The minuses andpluses a t the bottom of the figure indicate the treatment of each sample at each stage. Lunes 1 contain a-subunit overlaid with 3 pg of V8 protease, lanes 2 contain a-subunit that had been electrophoresed initially under the opposite condition, overlaid with 3 pg of V8 protease. Lanes 3,5, and 6 contain intact IgG (system A) or IgG heavy chain dimer (system B). Lune 4 (system A) contains

a-bungarotoxin binds to the V8-20 fragment and not to V8- 18.

The site of alkylation by [3H]meproadifen mustard was located on a peptide distinct from V8-20, designated V8-21. However, this peptide appeared similar to V8-20, since it contained the [3H]MBTA-reactive site when a-subunit was isolated from AChR reacted with nonradioactive meproadifen mustard and [3H]MBTA (Fig. 3). In addition, as for V8-20, the mobility of V8-21 was unaltered after treatment of the AChR with Endo H. Thus, the meproadifen mustard-reactive site is probably located within the peptide defined by V8-20.

FITC-ConA bound to the 18-kDa peptide beginning at a- subunit Val-46, V8-18, and the mobility of this fragment was sensitive to prior Endo H treatment of the a-subunit. These results are consistent with the presence of N-linked carbohydrate on V8-18 and in agreement with the proposed attachment site for N-linked carbohydrate at Asn-141 (Noda et al., 1982). In certain conditions, an incompletely cleaved form of this fragment co-migrates with V8-20. In these conditions, the N terminus of Val-46 was also found with V8-20, a result that suggests that the C terminus of this fragment was not completely trimmed.

The FITC-ConA binding results and the sequencing data together have significant bearing on a hypothesis proposed by Conti-Tronconi et al. (1984). These authors also sequenced what was probably the equivalent of the fragments we have named V8-20 and V8-18. They reported that both bands had N-terminal sequence beginning at Val-46. A comparison of periodic acid-Schiff base staining of these bands showed stronger staining of V8-18, indicating the presence of more carbohydrate on this band. These results, and the observa- tions by Gullick et al. (1981) that the MBTA-reactive site was contained in V8-20, led these authors to propose that the ability of MBTA to react preferentially with only one of the two a-subunits in the AChR (Damle and Karlin, 1978) was due to differential glycosylation of the two a-subunits.

The results presented here suggest that this hypothesis is incorrect. The sequencing results performed with or without Endo H treatment demonstrate that the [3H]MBTA-reactive site lies beyond Ser-173 and is not part of a peptide fragment beginning at Val-46. The results also show that, under certain conditions, a carbohydrate-containing peptide beginning at Val-46 will co-migrate with the [3H]MBTA-labeled peptide. Thus, the differential glycosylation of the two bands observed by Conti-Tronconi et al. (1984) may reflect the extent of cleavage by V8 protease, resulting in variable amounts of peptide with an N terminus at Val-46 in V8-20 and V8-18. The data presented here are also consistent with their determination of a contaminating sequence beginning at Val-46 that migrates as V8-20, although we cannot account for their failure to detect the sequence beginning at Ser-173. Further- more, the results of Endo H treatment demonstrate that there is no evidence for differential glycosylation of a-subunits based on proteolytic mapping with V8 protease.

Comparison of the V8-proteolytic pattern in the absence or presence of sulfhydryl-reducing agents revealed no differ-

IgG heavy chain dimer, excised from the first gel, run in system B. Lane 4 (system B) contains unreduced IgG isolated from the first gel, run in system A. Lunes 3 and 4 did not contain V8 protease. Lane 5 was overlaid with 3 pg of V8 protease. Lune 6 was overlaid with 3 pg of V8 protease that had been inactivated by boiling for 10 min. The guide at the bottom of the figure summarizes the presence of V8 protease (V8-P; b, boiled V8), the presence of reducing agent in the first (8% acrylamide) gel (SH-1) or the second (proteolytic cleavage) gel (SH-2). Migration of standards are indicated on the right and relevant bands are identified on the left. HL, intact IgG; H, IgG heavy chain; L, IgG light chain; P, V8 protease.


ences. Therefore, there were no disulfide linkages between cysteines contained in V8-18 to any cysteines contained in V8-20. The sequence determination and carbohydrate analysis of V8-18 and V8-20 predicts that V8-18 contained Cys- 128 and Cys-142 and that V8-20 contained Cys-192, Cys-193, and Cys-222. Thus, disulfide linkages from Cys-128 or Cys- 142 to Cys-192, Cys-193, and Cys-222 can be ruled out. This conclusion excludes several hypothesized models for the structure of the AChR a-subunit (Boulter et al., 1985; Kao et al., 1984; Criado et al., 1985) that involve disulfide bonding among these cysteines.

The data presented here indicate that only V8-20 contributes to the agonist-binding site, since all the ligands directed to the agonist-binding site that were tested, [3H]d-tubocurare, [3H]MBTA, and '251-a-bungarotoxin, interact only at this fragment. The analysis of the proteolytic pattern in the absence of reducing agent further suggests that the disulfide bond located in the vicinity of the ACh-binding site is not composed of any cysteines contained in V8-18. Thus, the part of the protein implicated as part of the ACh-binding site lies beyond Ser-173. Although other portions of the protein cannot be excluded as contributors to the ACh-binding site, there is no evidence to suggest that any portion outside of V8-20 contributes to the site.

The studies here provide an initial characterization of location of ligand-binding sites within the AChR a-subunit as well as the pattern of disulfide bonding within that subunit. Additional studies that are underway will serve to identify within the a-subunit primary structure the amino acids re- acting with the affinity labels for the ACh-binding site and the allosteric antagonist-binding site.

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D., Stengelin, S., Morton, G., Heinemann, S., and Patrick, J. (1985) J. Neurosci. 5, 2545-2552

Boyd, N. D., and Cohen, J. B. (1980) Biochemistry 19,5344-5353 Burridge, K. (1978) Methods Enzymol. 50,54-64 Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U.

K. (1977) J. Biol. Chem. 252,1102-1106 Cohen, J. B., Medynski, D. C., and Strnad, N. P. (1985) in Effects of

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Cox, R. N., Kaldany, R.-R. J., DiPaola, M., and Karlin, A. (1985) J. Biol. Chem. 260,7186-7193

Criado, M., Sarin, V., Fox, J. L., and Lindstrom, J. (1985) Biochem. Bwphys. Res. Commun. 128,864-871

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Dreyer, E. B. (1984) Ph.D. Dissertation, Harvard University 2321-2328

Dreyer, E. B., Hasan, F., Cohen, S. G., and Cohen, J. B. (1986) J.

Finer-Moore, J., and Stroud, R. M. (1984) Proc. Natl. Acud. Sci. U.

Gershoni, J. M., Hawrot, E., and Lentz, T. L. (1983) Proc. Natl. Acud.

Gullick, W. J., Tzartos, S., and Lindstrom, J. (1981) Biochemistry

Heidmann, T., Oswald, R. E., and Changeux, J.-P. (1983) Biochem-

Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J.

Hunkapiller, M. W., Lujon, E., Ostrander, F., and Hood, L. E. (1983)

Jeng, A. Y., St. John, P. A., and Cohen, J. B. (1981) Biochim. Biophys.

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Kao, P. N., and Karlin, A. (1986) J. BWZ. Chem. 261,8085-8088 Kao, P. N., Dwork, A. J., Kaldany, R.-R. J., Silver, M. L., Wideman,

J., Stein, S., and Karlin, A. (1984) J. Biol. Chem. 259, 11662- 11665

Biol. Chem. 261,13727-13734

S. A. 8 1 , 155-159

Sci. U. S. A. 80,4973-4977

20,2173-2180

istry 22,3112-3127

(1981) J. Bid. Chem. 256,7990-7997

Methods Enzymol. 9 1,227-236

Acta 646,411-421

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Laemmli, U. K. (1970) Nature 227, 680-685 Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56,335-341 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)

J. Biol. Chem. 193 , 265-275 McCarthy, M. P., Earnest, J. P., Young, E. F., Choe, S., and Stroud,

R. M. (1986) Annu. Reu. Neurosci. 9,383-413 Neher, E. (1983) J. Physwl. (Lord.) 339,663-678 Neubig, R. R., and Cohen, J. B. (1979) Biochemistry 18,5464-5475 Neubig, R. R., Krodel, E. K., Boyd, N. D., and Cohen, J. B. (1979)

Proc. Natl. Acud. Sci. U. S. A. 76, 690-694 Neumann, D., Gershoni, J. M., Fridkin, M., and Fuchs, S. (1985)

Proc. Natl. Acud. Sci. U. S. A. 8 2 , 3490-3493 Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furatami, Y.,

Nature 2 9 9 , 793-797 Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982)

Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Hirose, T., Asai, M., Takoshima, H., Inayama, S., Miyata, T., and Numa, S. (1983a) Nature 310 , 251-255

Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutami, Y., Hirose, T., Tahashima, H., Inayama, S., Miyata, T., and Numa, S. (198313) Nature 302,528-532

Oswald, R. E., and Changeux, J.-P. (1981a) Proc. Natl. Acud. Sci. U.

Oswald, R. E., and Changeux, J.-P. (1981b) Biochemistry 2 0 , 7166-

Peper, K., Bradley, R. J., and Dreyer, F. (1982) Physiol. Reu. 6 2 ,

Popot, J.-L., and Changeux, J.-P. (1984) Physiol. Reu. 64,1162-1239 Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 5 6 , 502-

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Wilson, P. T., Lentz, T. L., and Hawrot, E. (1985) Proc. Natl. Acud.

Wolosin, J. M., Lyddiatt, A., Dolly, J. O., and Barnard, E. A. (1980)

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Materials: Receptor-rich membranes were isolated from the electric organ of Torpedo californlca according to the procedure of Sobel et a l . (1977). except that fresh electrlc organ was homogenized in the p r e s e n c m 0 "g leupeptin. 10 mg pepsratin, 10 mg aprotinin. 18 mg phenylmethylsulfonyl fluoride. and -02% NaN I n I I H20. ,All eubsequent Solutions contained ,028 NaN The final me%brane suspension was stored at -80.C under argon and typi2;lly contained 1-2 nmol ACh binding sites per mg protein. as measured by a d i r e c t acetylcholine b i n d l n g assay (Boyd and Cohen, 19801. Membranes from T. rnarrnorata were provided by Prof. J.P. Changeui (Pasteur tnstltute). -

grade) was from Plerce. Zetabind blottlng paper was from RHFICUNO. Carbanyl- choline and d-tubocurare chloride were from Sigma. u-Bunqarotoxin was from Miami Serpenrariurn. Other reagents were from standard sources.

, ~ . ~ ~ ~ ~ . .

Affinity labelrng praSeduceo: The u-subunlt of the acetylcholine receptor was trace labelled with L<HJMBTA using the procedure of Wolosin et al. 11980). Receptor-rich membranes were centrifuged and resuspended in NaC1/4.5 mM NaN 11 5 mM EDTRjl5 mM T r i s - I I C 1 pH 8.0. Reduction was carried o u t by the add!t i& of dithiokhreitol ( D T T ) to 0 . 3 WN final concentration and incubation for 3 0 min at ambient temperature. The membrane8 Yere collected by centrifugation LO relnove DTT and resuspended in Tor edo physiological saline (250 mH NaC115 zp K C 1 / 3 mM C a C l 12 mM H g C l 5 mM sodlvm phosphate, pH 1.01 0.02% NaN3:

bated for 5 mln. The incubation was Stopped by the additLon of 2-mercapto- TPS). HIMBTA wae add,$ to the m e d r a n e ~ c i per mg proteln) and I ~ C U -

ethanol to 14 mM final c ncentratron. The labelled membranes were centrifuged again to remove exce68 [qH3MBTA and then solubilized for electrophoresis.

Up LO 25% of the added [3HIM8TA was lncorporared. No labelling of any pro. ryplcally. le33 than 2% of the receptors were labelled by this procedure whrle

telns except the n-subunit of the AChR wag detected by fluorography.

Labellinq of membranes with [3Hld-tubocurare was carried out bv the eXpasure of AChR-TiCh membranes to UV light in the presence of the i a&l a s descrrbed by E. 8 . Dreyer 11984). Membranes were centrlfuged and resuspended I n TPS to a concentration of 4 mq per ml. [IHld-tubocurare wag added to 5 V M and the mixture flushed with A r far 3 0 min. The mixture wag then exposed to UV llght IMinerallght, model UVSL25. Ultraviolet Products Inc.) for 10 m z n from a dlstance of 10 m . The membranes were col lected by centrifugation for LO min ~n a Beckman Airfuge at 28 PSI and solubilized far gel electrophoresis.

carried out essentially as described in the accompanying paper (Dreyer ee al., Labelling of membranes with l3illrneproadifen mustard (6 Cilmol) was

19861. wlth several modifrcations. Labelling was carrled out at a m b i e n i - temperature l22'C) for 30 mi" in alrfuge tubes. The [3ri]rneproadlfen mustard concentration was 20 iM durlng labelllng, in the presence of 3 mM carbamyl- chaline. Nonspecific labelilng was determined by the e x t e n t of labelling i n the presence of 200 uM meproadifen. Thls proceclure apparently ylelde3 optimal I belling. which vas usually about 15% Of the AChR labelled specifically by

dilution wlth 150 rl 10 mM sodium thiosulfate and membranes were then col- ['Hlmeproadrfen mustard. After labelllng. the reaction w a g quenched by

1ecteO by centrlfvgation in the Airfuge and prepared for electrophoresis.

electrophoresle utilizing a Savant preparative gel c e l l (model PAC 15WC). The Is01dtl0n of o-subunit: Pure a-subunit was isolated by preparative tube gel

43 kDa non-receptor protein Of the nicatrnic post-synaptic membranes was first

order to lrnprove the yield of pure u-suhunit. After alkalineextract~on. removed from the membranes by alkaline extraction (Neubig et a l . . 1479) in

menbranee were Coltected by centrlfugacion and resuspended in buffer appro- p r ~ a t e for the labelling protocol to be used (if any). After la hell in^, the

v lde cylindrlcal gel containing an 8-cm separailng vel 18% acrylarnide) and a sample was dissolved for electrophoresis. The sample was loaded onto a 1.5-cm

night at 15 mA. Proteins eluting from the gel via a flow chamber were cal - 2-cm long stackrng gel (4% acrylamldel. Electrophoresis w a s carried out over-

Cians were dasayed by SDS-PAGE ( s e e beloul. and the fractions containing pure lected as 1 rnl fractions in 10 mM Tris-nC1, pH 8.011 mM m'r/O.l% SDS. Frac-

a-subunit were pooled. Typically, about 500 ug of pure u-subunit could be recovered from 12 mg of AChR-rich membranes. Pure a-subunit was stored i n small ailquois at -80.C under a r g o n .

Gel electrophoresis: Electrophoresis was carried out u51ng the proceciure descrlbed by L a e m i 11970). For separating the subunits of the kChR the resolving gel Was :% dcrylarnlde wlth 0.32% crosrlrnker and the stack~iq gel was 4% with 0.16% crosslinker. Slab g e l s ( 1 4 x 1 6 cm) were usually 1.5 mm thick. Samples were dissolved in Iaernmli sample buffer 160 mM T r l s - ~ C l , pH

o x l d a t l o n of proteins during electrophoresis. 0.2 n M sodium thinglycolate was 2-rnercaptoethanol vas excluded from the sample buffer. In order to prevent

included in elecrrade buffer Iliunkapiller et al.. 19831. u n l e s s SpeCiflcdlly

8 mA for overnight runs. After electrophoresis the gels were fixed in 50% noted otherwise. Electrophoresis w a g performed at 20-30 mA for 5-6 hrs or at

merhanol/lO% acetic a c l d for 30 mln and then s t b ~ n e a wlrh Coomassle Brilliant

6.812% sos/8% sucrose118 glycerol/2% 2-mercaptaethanoll. xn some i n s t a n c e s ,

acetic acid) for 1 hr. Higher percentage aciylamide gels were stained l o n g e r . ~ l u e solution (0.25% coomassie Brilliant ~ l u e R (sigma11 45% methanol1108

The gels were destained with 25% methanol1108 acetic acid until bands were optimally visible (1-20 hre).

Proteolytrc mapp~ng was performed uning the method of Cleveland (1977). using s . aureus "8 protease. The proteolytic cleavage takes place during the stacking phase of electrophoresis in the Laemmli gel system. TO allow sufficient time for cleavage and to ensure complete stackxng. the stack- in. ael was exceDtionallv lono. 5 cm. Stackina and or0Leo1~s1s were carrred

TWO distinct proceduree for loading the samples in the proteolytic mdp- ping g e l s were used. Procedure A: The membranes were labelled by one or more of the procedvres described above. collected by centrifugation in the Rirfllge

solved f r o m Other proteins using am 8% slab gel as deacribed above. The Pro- (28 PSI), and then dissolved in Laemli sample buffer. The u-aubunrt was r e -

tein bands were vleuallred by staining the gel for 20 to 30 mi" with Coomassie Brilliant Blue without preflirng. The band contalnlng the o-subunit waa excised and soaked for 3 0 to 60 min in 5% sucrose/l25 mM Tria-HC1. pH 6.810.18 SDS loverlav buffer1 olus 1 mM DTT. Each slice was then transferred to a well

pararive tube gel electrophoresia, as described above, was concentrated 5 to . .

10-fold using a Centricon miCrOcOncentratDr (Amicon) with a 30 kDa cutoff membrane and then mlxed with a 5x concentrate Of sample buffer to yleld the correct final concentration of sample buffer. 10 u l of 0.2 m g / m l VB-protease was also added where required. The entlre sample was irmnedlately loaded into the well of the mapping gel and electrophoresed as deecribed above.

Radiolabelled proteins and peptides that had been separated by gel electrophoresis were detected by fluorography. After Coomassie Blue staining of the gel, the gel was placed in Enhance INEN) for 1 hr, then soaked for 1 hr in

prevent cracking) and exposed to Kodak XAR-5 film. The film was preflaahed as 1% glYcerol/5% acetic acid. The gel was dried at low temperature (70.C. to

described by Laskey and H i l l s (1975). Exposure times are given in xndlvidual figure legends.

Ancillary Procedures: PrOtrln was measured by the method of Loury et al. 11951) or by the method of Schaffner and Peissmann 11973). __

location of ligand-binding sites on the nicotinic ... · location of ligand-binding sites on the...

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