Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2553-2557, April 1984 Neurobiology Binding of a-bungarotoxin to proteolytic fragments of the a subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters (electrophoretic transfer/maleimido-benzyltrimethylammonium/peptide map/protease/receptor-ligand interaction) PAUL T. WILSON*, JONATHAN M. GERSHONI*t, EDWARD HAWROTt, AND THOMAS L. LENTZ* *Departments of Cell Biology and 4Pharmacology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06510 Communicated by Charles F. Stevens, December 28, 1983 ABSTRACT Proteolytic fragments of the a subunit of the could be demonstrated to the a subunit but not to the other acetylcholine receptor retain the ability to bind a-bungaro- chains. Further dissection of the receptor can be achieved toxin following resolution by polyacrylamide gel electrophore- through the preparation of peptide fragments by limited pro- sis and immobilization on protein transfers. The a subunit of teolysis of isolated subunits. This approach has been em- the acetylcholine receptor of Torpedo electric organ was digest- ployed, for example, to compare the subunit structures of ed with four proteases: Staphylococcus aureus V-8 protease, AcChoR from different sources (11, 12) and to characterize papain, bromelain, and proteinase K. The proteolytic frag- the domain specificity of anti-AcChoR monoclonal antibod- ments resolved on 15% polyacrylamide gels were electropho- ies (13). One difficulty in utilizing parts of the AcChoR to retically transferred onto positively charged nylon membrane study receptor function is that as the AcChoR is dismantled filters. When incubated with 0.3 nM 1251-labeled a-bungaro- its characteristic functions may become less recognizable or toxin and autoradiographed, the transfers yielded patterns of no longer detectable. However, if small fragments of the Ac- labeled bands characteristic for each protease. The molecular ChoR that retain essential functions can be identified, corre- masses of the fragments binding toxin ranged from 7 to 34 lation of the structural characteristics responsible for these kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. functions would be facilitated. The apparent affinity of the fragments for a-bungarotoxin as An important tool in the investigation of AcChoR has been determined from the IC50 value was 6.7 X 10-8 M. The label- the use of snake venom a-neurotoxins, such as a-BTX, ing of fragments with a-bungarotoxin could be inhibited by which block the binding of cholinergic ligands to the receptor prior affinity alkylation of receptor-containing membranes and thus are believed to bind at or near the AcCho binding with 4-(N-maleimido)-a-benzyltrimethylammonium iodide. site on the a subunit (14). These ligands that bind with a high These findings demonstrate that immobilized proteolytic frag- degree of specificity and affinity can be used as probes to ments as small as 1/5 the size of the a subunit retain the struc- characterize the nature of receptor binding sites. Recently, tural characteristics necessary for binding a-bungarotoxin, al- we have introduced a novel approach in studying the molec- though the toxin is bound to the fragments with lower affinity ular aspects of such receptor-ligand interactions (15). Pro- than to the native receptor. The effect of affinity ligand alkyl- tein blotting or electrophoretic transfer of dissociated pro- ation demonstrates that the a-bungarotoxin binding site de- teins resolved by polyacrylamide gel electrophoresis onto tected on the proteolytic fragments is the same as the affinity- immobilizing matrices has permitted the direct demonstra- labeled acetylcholine binding site on the intact acetylcholine tion of various protein-ligand interactions, such as antibod- receptor. ies with antigens and lectins with glycoproteins (for review, see ref. 16). Protein blotting has been generally employed as The nicotinic acetylcholine receptor (AcChoR) that func- a qualitative assay; however, we have refined this technique tions to transduce a chemical signal into an electrical event to a quantitative assay of protein-ligand association. In this in many neuronal systems has been well characterized both manner, the binding of a-BTX to the isolated a subunit of the physiologically and biochemically (for review, see refs. 1 AcChoR of Torpedo was demonstrated directly and the ki- and 2). The AcChoR purified from the electroplaque of the netics and characteristics of the interaction were investigat- electric ray Torpedo californica consists of four subunits ed (15). Furthermore, it could be demonstrated that the bind- present in the molar stoichiometry of a2/3y8 (3-5). This pen- ing of a-BTX is specific and close to the physiologically rele- tameric complex contains the agonist binding sites responsi- vant AcCho binding site on the receptor as It was inhibited ble for receptor activation and desensitization as well as the by the cholinergic antagonist d-tubocurarine and the affinity ion channel for cation flux through the membrane. Recent alkylating agent 4-(N-maleimido)-a-benzyltrimethylammoni- information on the primary amino acid sequences of the um iodide (MBTA). polypeptides comprising the receptor (5-9) provides a basis In the present study, in an effort to gain further informa- for analyzing and understanding these receptor functions at tion on the nature of the AcCho binding site on the AcChoR, the molecular level. we have extended the protein transfer approach and ana- One approach in elucidating receptor function is the dis- lyzed a-BTX binding to isolated, immobilized peptide frag- mantling of the intact receptor into its constituent parts and ments prepared by proteolytic digestion of the a subunit of determination of the functions and properties of these sim- pler components. For example, the four subunits of the Ac- T. californica AcChoR. It is demonstrated that protpolytic ChoR have been isolated by gel electrophoresis in Na- fragments of the a subunit can be separated and immobilized DodSO4 and tested for a-bungarotoxin (a-BTX) binding af- ter removal of the NaDodSO4 (10). Saturable a-BTX binding Abbreviations: AcCho, acetylcholine; AcChoR, AcCho receptor; a- BTX, a-bungarotoxin; LiDodSO4, lithium dodecyl sulfate; MBTA, 4-(N-maleimido)-a-benzyltrimethylammonium iodide; PCM, posi- The publication costs of this article were defrayed in part by page charge tively charged membrane. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Biophysics, Weizmann Institute of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Science, Rehovot, Israel. 2553 2554 Neurobiology: Wilson et al. Proc. NatL Acad Sci. USA 81 (1984) and that these fragments retain a-BTX-binding characteris- in, S. aureus V-8 protease, or bromelain. The fragments tics comparable to those of the intact a subunit. Preliminary were separated by gel electrophoresis, transferred, and incu- reports of some of this work have appeared (17, 18). bated with 125I-labeled a-BTX, and the amount of toxin bound to fragments and intact a subunit was determined by autoradiography and quantitation of bands as described MATERIALS AND METHODS above. Preparation of Torpedo Electric Organ Membranes. Mem- branes were prepared from frozen electric organ tissue of T. RESULTS californica (Pacific Biomarine, Venice, CA) as described 125I-labeled a-BTX was found to bind on protein blots to sev- (15). Based upon the number of a-BTX binding sites as de- eral peptide fragments generated by proteolysis of the a sub- termined by an immunoprecipitation assay (15), the AcChoR unit of Torpedo AcChoR. Digestion of the a subunit excised comprised about 5% of the total protein of this preparation. from LiDodSO4/10% polyacrylamide gels with various pro- Gel Electrophoresis and Proteolytic Digestion. Torpedo teases generated peptide fragments that were resolved on electric organ membrane was solubilized at room tempera- NaDodSO4/15% polyacrylamide gels and subsequently elec- ture for 1 hr in 50 mM Tris HCI, pH 6.8/3% (vol/vol) 2- troeluted onto PCM filters. After transfer, the filters were mercaptoethanol/2% (wt/vol) lithium dodecyl sulfate (Li- processed and incubated with 125I-labeled a-BTX. In the DodSO4; Sigma)/0.1% bromophenol blue. The sample was case of untreated a subunit, after autoradiography a single then resolved on a 10% polyacrylamide gel as described (19), radioactive band at an electrophoretic mobility correspond- except that EDTA was omitted from the upper reservoir ing to -40 kDa was detected (Fig. 1, lane A). After digestion buffer. Electrophoresis was carried out at 40C at constant by S. aureus V-8 protease (Fig. 1, lane B), papain (Fig. 1, current (20 mA) overnight. Protein bands were visualized by lane C), bromelain (Fig. 1, lane D), and proteinase K (Fig. 1, placing the gel in 4 M sodium acetate as described (20). The lane E), a pattern of bands was obtained that was consistent band representing the a subunit of the AcChoR was excised and highly reproducible for each protease. Digestion with S. from the gel and placed into a solution of 125 mM Tris HCl, aureus V-8 protease resulted in three areas that bound 1251. pH 6.8/0.1% (wt/vol) NaDodSO4 for 30 min at room tem- labeled a-BTX: single bands at mobilities corresponding to perature. Gel slices were then placed into the wells of a -32 kDa and -27 kDa and a closely spaced, major group of NaDodSO4/15% polyacrylamide gel and digested in the bands (triplet) at 18-19 kDa. With extensive digestion, a stacking gel with either Staphylococcus aureus V-8 protease band at 9 kDa was observed. Proteolysis with papain pro- (Miles Laboratories), papain (Sigma), bromelain (Sigma), or duced a more complex pattern with bands at mobilities of proteinase K (Merck) by the procedure of Cleveland et al. -33-34, 28, 25, 16-17, 10, and 7 kDa. Bromelain treatment (21), except for the omission of EDTA from all solutions.