0736-5748/85 $03.00+0.00 Pergamon Press Ltd. © 1985 ISDN

Int. J. Devl. Neuroscience, Vol. 3, No. 2, pp. 123-134, 1985. Printed in Great Britain.

NICOTINIC A C E T Y L C H O L I N E R E C E P T O R F R O M FOETAL HUMAN SKELETAL MUSCLE GILLIAN M. TURNBULL, ROGER HARRISON and GEORGE G. LUNT Department of Biochemistry, School of Biological Sciences, University of Bath, Claverton Down, Bath BA2 7AY, U.K. (Accepted 16 July 1984)

Abstract--Nicotinic acetylcholine receptor protein has been purified from foetal and adult skeletal muscle by extraction in non-ionic detergent followed by purification on immobilised a-toxin. Purified foetal and adult receptors focused as single, sharp peaks whether directly labelled with t251or indirectly labelled with a251-a-bungarotoxin. Polyacrylamide gel electrophoresis of the purified foetal and adult receptors each showed four major protein bands with M, 44,000, 51,000, 58.000 and 66,000; only that with M,. 44,000 was, in each case, labelled with the affinity reagent, 4-(N-maleimido) [3H]benzyltrimethylammonium. When the four major subunits, obtained by polyacrylamide gel electrophoresis, were labelled with ~2Sl-ConA,markedly different patterns of radioactivity were shown by the foetal and adult receptors, the band at 44Jl00 being less heavily labelled in the foetal case. Foetal and adult receptors behaved similarly with respect to inhibition by ConA of binding of ~2Sl-c~-bungarotoxin;inhibition in both cases reaching a maximum of 70%. Foetal and adult receptors each showed single t251-e~-bungarotoxin binding species in sucrose density gradient centrifugation with s2o,,=8.5S and 9.5S, respectively. although the former peak was broader, possibly reflecting the relative instability of the purified foetal receptor. Our finding of marked differences in the glycosylation of foetal and adult human acetylcholine receptors suggest that. in otherwise very similar proteins, the carbohydrate moieties could determine the known differences in location and stability of the two receptor types. Key words: Foetal human muscle, Nicotinic acetylcholine receptor, Carbohydrate.

T h e nicotinic a c e t y l c h o l i n e r e c e p t o r f r o m the electric o r g a n s of v a r i o u s species of fish has b e e n i s o l a t e d a n d c h a r a c t e r i s e d to the e x t e n t that the a m i n o acid s e q u e n c e of the c o n s t i t u e n t subunits is k n o w n . 27 F u r t h e r m o r e the purified p r o t e i n has b e e n r e c o n s t i t u t e d in a f u n c t i o n a l state in artificial m e m b r a n e s . 22 T h e m e t h o d o l o g y t h a t has b e e n a c q u i r e d in t h e s e studies has b e e n a p p l i e d to the m o r e difficult p r o b l e m o f isolating the r e c e p t o r from m a m m a l i a n muscle a n d this has r e s u l t e d in the purification a n d p a r t i a l c h a r a c t e r i s a t i o n of r e c e p t o r p r o t e i n s f r o m a v a r i e t y o f m a m m a l i a n sources2.~.l 1.12,15.23,25,26.32.33 including h u m a n muscle, e5'33 F r o m these studies a c o m m o n p a t t e r n is e m e r g i n g in that the r e c e p t o r p r o t e i n from all s o u r c e s a p p e a r s to c o n t a i n s o m e o r all o f the s a m e f o u r s u b u n i t s c~, [3, ~,, ~, that a r e c h a r a c t e r i s t i c of the fish r e c e p t o r s . 5 I n d e e d , r e c e n t cloning es of calf c D N A a n d h u m a n g e n o m i c D N A c o d i n g for the a - s u b u n i t p r e c u r s o r o f m u s c l e r e c e p t o r has led to the d e m o n s t r a t i o n that the a m i n o acid s e q u e n c e s of b o t h m a m m a l i a n f o r m s show m a r k e d h o m o l o g y with the Torpedo r e c e p t o r . A c e t y l c h o l i n e r e c e p t o r s of e m b r y o n i c muscle are k n o w n to differ in a n u m b e r of ways f r o m t h o s e o f n o r m a l muscle. W h e r e a s the l a t t e r are r e l a t i v e l y stable a n d confined to the s y n a p t i c r e g i o n , e m b r y o n i c r e c e p t o r s are d i s t r i b u t e d o v e r the m u s c l e surface, show v a r y i n g d e g r e e s of l a t e r a l m o b i l i t y a n d have f a s t e r t u r n o v e r rates; p r o p e r t i e s which are also a s s u m e d by a d u l t r e c e p tors f o l l o w i n g d e n e r v a t i o n . 9 A l t h o u g h it m i g h t be e x p e c t e d that such d i f f e r e n c e s b e t w e e n ext r a j u n c t i o n a l a n d j u n c t i o n a l rec,:ptors w o u l d be r e f l e c t e d in t h e i r m o l e c u l a r s t r u c t u r e s , e v i d e n c e for this is s p a r s e a n d largely d e r i v e d from d e n e r v a t e d muscle. T h u s , a c e t y l c h o l i n e r e c e p t o r s o f a d u l t n o r m a l a n d d e n e r v a t e d rats have b e e n r e p o r t e d to have slightly d i f f e r e n t isoelectric p o i n t s 4 and to differ in t h e i r i n t e r a c t i o n s with a n t i - r e c e p t o r a n t i b o d i e s in the s e r a o f m y a s t h e n i c p a t i e n t s ; 14,31,38 similar a n t i g e n i c d i f f e r e n c e s b e t w e e n n o r m a l a n d d e n e r v a t e d a d u l t h u m a n muscle have also b e e n reported.17'36 Less c o m p a r a t i v e d a t a are a v a i l a b l e for foetal r e c e p t o r s . In t h e i r assays using m y a s t h e n i c sera, W e i n b e r g a n d Hall 38 s h o w e d that a c e t y l c h o l i n e r e c e p t o r from 18d a y - o l d rat e m b r y o s was a n t i g e n i c a l l y d i f f e r e n t f r o m that o f n o r m a l a d u l t rats. O n the o t h e r h a n d , L o t w i c k et al.2" w e r e u n a b l e to d e t e c t similar d i f f e r e n c e s for h u m a n r e c e p t o r s and S u m i k a w a et Abbreviations: Sodium dodecyl sulphate; TEMED, NNNN'-tetramethylethylenediamine; DNase, deoxyribonuclease; C¢,nA, Concanavalin A.

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al. :~4:~5 could not distinguish bctwecn embryonic and adult chicken muscle receptor by either biochemical or immunological assays. Possible ,~tructural differences between the embryonic and adult forms of the acetytcholinc receptor are rclevant not only to its functional development but also to its rccogniscd role as the autoimmunogcn in myasthenia gravis. ~~ Wc havc, accordingly, purified the human foetal rcccptot and hcre present a comparison of its propcrtics with those of the adult form. M A T E R I A L S AND M E T H O D S Materials

c~-Bungarotoxin was obtained from Boehringer Corporation (Lewes, Sussex, U.K.). Naja naja siamensis venom was from the Miami Serpentarium (Miami, FL, U.S.A.). Carrier-free Na 1251in dilute N a O H solution (100 mCi/ml) was from the Radiochemical Centre, Amersham, Bucks, U.K. and benzoquinonium chloride was a generous gift from Stifling Winthrop Inc., Rensselaer, NY, U.S.A. Gel filtration reagents were supplied by Pharmacia Ltd., Hounslow, U.K. Ampholines pH 5-7, pH 3.5-10 were from LKB Ltd., Croydon, U.K. All other materials were from Sigma Chemical Co., Kingston-upon-Thames, U.K. or from BDH Chemicals, Poole, Dorset, U.K. Methods

A modification of the method of Stephenson et al. 33 was used. Adult human muscle was obtained from leg amputations carried out because of either vascular abnormality or diabetic gangrene. In the latter case only those limbs in which the gangrene was confined to the toes was used. The muscle, mainly gastrocnemius was coarsely chopped and frozen immediately in liquid nitrogen. For the purification of foetal human muscle, foetuses (1222 weeks) obtained from prostaglandin-induced terminations were frozen to -80°C within 5 h. Muscle was removed from the back, chest and limbs and was coarsely chopped prior to the extraction as described previously. 33 Unless otherwise stated, all operations were carried out at 4°C and buffers throughout contained 1 mM phenylmethyl sulphonyl fluoride, 0.02% (w/v) NAN3, 1 mM E D T A at pH 7.4. Muscle (200-300 g) was homogenised for 1 min at full speed in a Waring blender or Sorvall Omnimix in 10 mM potassium phosphate buffer (5 vol.) containing 50 mM NaCI, 1 mM benzamidine hydrochloride, 0.1 mM benzethonium chloride, soyabean trypsin inhibitor (10 ~xg/ml), bacitracin (100 p,g/ml) and, where stated, 10 mM iodoacetamide. After centrifugation at 20,000 g for 1 h, the pellets were homogenised for 2 x 1 rain at full speed in the above buffer (1-2 vol.) containing 2% (v/v) Triton X-100, and were extracted by stirring for 2-3 h either at 4 or 23°C. The extract was centrifuged for 1 h at 100,000 g and any fat aspirated from the supernatant. The supernatant (crude receptor preparation) was stirred for 4 h with Naja naja siamensis c~-toxin coupled to Sepharose 4B ~'21 (25 ml packed volume carrying 12.5 mg bound c~-toxin), either at 4 or 23°C. The affinity beads were filter washed with 10 mM potassium phosphate buffer containing 0.1% (v/v) Triton X-100 (buffer B), with buffer B containing 0.5 M NaCI (1 1) and then with buffer B alone (1 1). Acetylcholine receptor was eluted from the c~-toxin affinity beads by 2 methods. In the first method elution was by batchwise stirring with 1 M carbamoylcholine in buffer B (50 ml) for 4 h or overnight, the eluate being subsequently dialysed against buffer B (5 1) with E D T A omitted. The dialysate was applied to a D E A E cellulose column (2 × 2 cm) and extensively washed with buffer B (1 1) following which the receptor was eluted with 0.5 M NaCI in buffer B (20 ml). The alternative elution method involved packing the washed c~-toxin Sepharose 4B beads into a column (2 × 8 cm), eluting the receptor by passing 3 mM benzoquinonium chloride in buffer B (30 ml) through the affinity column directly onto a D E A E cellulose column (2 x 2 cm) and recycling for 16 h. The D E A E cellulose column was washed with buffer B (100 ml) and eluted as described above. One further purification step used, where stated, involved stirring the purified receptor with DNase 1 Sepharose 4B (0.1 ml packed gel/ml acetylcholine receptor) for 2 h at 4°C and then filtering.

Foetal human acetylcholine receptor

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Assay of acetylcholine receptor Crude detergent extracts and purified acetylcholine receptor were assayed as described previously. 33

lodination of acetylcholine receptor In dilute NaOH solution, carrier-free Na 1251(100 mCi/mi) (10 p~i) was added to up to 1 mg of receptor in 0.05 M potassium phosphate buffer pH 7.5. Chloramine T, 0.02% (w/v), in 0.05 M potassium phosphate buffer (125 ~1) was added and the mixture stirred over ice for 5 min, after which 0.02% (w/v) sodium metabisulphite in 0.05 M potassium phosphate buffer (125 I~1) was added. The mixture was chromatographed on a column of Sephadex G25 (25 × 1 cm) equilibrated in 0.01 M potassium phosphate buffer containing 0.1% (w/v) gelatin and 0.1% (v/v) Triton X100. Fractions were collected (20 × 1 ml) and the radioactive protein peak was identified by counting aliquots (5 I~1) of each fraction in an LKB 1280 Ultrogamma counter.

Assessment of antigenicity retained by acetylcholine receptor after iodination 125I-Acetylcholine receptor (100 ~l) was incubated with either normal human serum (5 ~1) or myasthenic serum (5 ~l) for 2 h at 23°C or overnight at 4°C in the presence of increasing concentrations of unlabelled acetylcholine receptor (0-1.0 nM). Goat anti-human IgG (60 ~1) was added and incubated as above, and the resulting precipitate was separated by centrifugation at 3000 g for 10 min. The pellet was washed with 10 mM potassium phosphate buffer containing 0.15 M NaCI, 1% (v/v) Triton X-100, 0.1% (w/v) NaN3 (0.5 mi) and centrifuged as above. The washing procedure was repeated three times, and the final pellet was counted for radioactivity using an EKB 1280 Ultrogamma counter.

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis under denaturing conditions was as described by Weber and Osborne 37 using disc gels (10 × 0.5 cm). After electrophoresis, gels were stained by using either Coomassie G250 or silver stain. Alternatively gels were frozen to -80°C, cut into l mm slices and counted with a LKB 1280 Ultrogamma counter.

Silver staining Gels were stained by using the method of Wray et al. 4° with modifications for the staining of disc gels. All solutions and washes contained double distilled water. Gels were soaked overnight in 50% (v/v) reagent grade methanol, with at least two changes, before being washed in water for a minimum of 20 min with one change. The staining solution was made by the dropwise addition of silver nitrate (0.4 g, 2 ml) to 0.36% (w/v) NaOH (10.5 ml) containing 14.8 M ammonium hydroxide (0.7 ml), with constant mixing. The volume of staining solution was made up to 50 ml, and gels were stained for 45 min with continuous mixing. After washing with water for 20 min, stain was developed by a solution (500 mi) containing 1% (w/v) citric acid (2.5 ml) and 38% (v/v) formaldehyde (0.25 ml). Bands appeared within 10 min and the development was stopped by immersion of the gel in 45% (v/v) methanol, 10% (v/v) glacial acetic acid.

Labelling with t25I-ConA ConA (1 mg) was iodinated as previously described for receptor. After normal fixing and/or staining, disc gels were extensively washed with continuous mixing in 0.5 M Tris-HCI pH 7.5 containing 0.15 M NaCI, 0.1% (w/v) NAN3, 0.5 mM CaC12 and 0.5 mM MgCI2. Gels were mixed in a solution of 125I-ConA in the above buffer (0.01q).04 Ci/mmol) for at least 4 h. After extensive washing in the same buffer, gels were frozen (-70°C), sliced and counted by using an LKB 1280 UItrogamma counter.

Affinity labelling of purified acetylcholine receptor Purified acetylcholine receptor was labelled with 4-(N-maleimido) [3H]benzyltrimethylammonium according to the method of Rubsamen et al. 3° as outlined previously. 33 DN 3 : 2 - B

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lsoelectric Jocusing lsoelectric focusing was performed on disc gels (20.5 cm × 1.5 mm) consisting of 5% (v/v) acrylamide, 1% (v/v) Triton X-100, 7% (v/v) ampholines (pH 5-7, pH 3.5-10), 0.2% (v/v) T E M E D and 0.2% (w/v) ammonium persulphate. The gels were pre-run at 400 V for 1 h with an anodal buffer of 0.01 M N a O H containing 1% (v/v) Triton X-100, pH 10.6, and a cathodal buffer of 0.4% (v/v) H2S04 containing 1% (v/v) Triton X-100. Samples were either iodionated receptor or were purified acetylcholine receptor (approximately 0.5-1.0 pmol) incubated with 0.5-1.5 nM L>l-c~-bungarotoxin (20 p~l) in the presence and absence of 0.01 M benzoquinonium chloride (20 ~1) for 90 rain at 23°C. Samples were focused at 1 kV for 6 h at 23°C. Gels were removed, sliced manually at 5 or 2.5 mm intervals and suspended in double distilled water (400 ~1) in stoppered tubes overnight at 23°C. Both the radioactivity and pH of each slice were measured.

Analytical ultracentrifugation of acetylcholine receptor Density gradient centrifugation was performed in linear sucrose gradients (4-20% w/v) in 10 mM potassium phosphate buffer pH 7.4 containing 0.1 M NaCI and 0.5% (v/v) Triton X-100. Velocity sedimentation was done in the SW 50.1 rotor of a Beckman L5-50B ultracentrifuge at 200,000 g (av) for 6 h at 23°C. Gradients were calibrated with [3-galactosidase (16.0S) catalase (11.4S) and yeast alcohol dehydrogenase (7.4S).

Inhibition by ConA of el-bungarotoxin binding to acetylcholine receptor The method used was that described by Wonnacott et al. 39 Purified acetylcholine receptor (100 ~xl) in 10 mM potassium phosphate buffer containing 0.1% (v/v) Triton X-100 and 0.1% (w/v) BSA was incubated for 30 min at 23°C with 0.02-1.0 mg/ml ConA (50 p,l). 125I-a-bungarotoxin (50 p~l, approximately 0.25 pmol) was added, incubated for 90 min and then filtered as for the D E A E cellulose assay. 33 As a control, 100 mM o~-methyl mannoside (50 p,l) was included during the pre-incubation.

RESULTS All experiments were performed 5 times unless otherwise stated; results are mean values _+S.E.

Purification of" acetylcholine receptor Detergent extraction of foetal muscle gave higher and more consistent 1251-a-bungarotoxin binding activities (0.99_+0.14 pmol/g tissue; n = 16) than did extraction of adult limb muscle (0.66 _+0.39 pmol/g; n = 10). The recovery of foetal acetylcholine receptor was similar to that previously described for adult skeletal muscle 33 representing approximately 20% of the detergentextracted activity and remained unchanged whether receptor was eluted from the a-neurotoxin affinity beads with carbamoylcholine or with benzoquinonium chloride. The ~25I-c~-bungarotoxin binding activity of foetal acetylcholine receptor is very unstable in contrast to that of the adult receptor, which remains unchanged at 4°C for several weeks. A crude extract of foetal muscle lost up to 30% of its original toxin binding activity within 48 h while affinity-purified receptor lost up to 80% of the original toxin binding activity within the same period (Fig. 1). Addition of neither 10% glycerol nor 2.5 mM phosphatidyicholine to purified acetylcholine receptor reduced this loss of activity.

lodination o¢"purified acetylcholine receptor Purified acetylcholine receptor was iodinated within 2 h of its elution from the DEAE-column and routinely had specific radioactivity of 20-40 Ci/mmol. In competition experiments (Materials and Methods section) specific binding of 12SI-labelled foetal acetylcholine receptor (200 fmol) by an excess of myasthenic serum was reduced to 50% by addition of an equivalent amount of unlabelled foetal receptor indicating that the radioiodination procedure had not caused any loss of antigenicity.

Foetal human acetylcholine receptor

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Fig. 1. Loss of ~251-tx-bungarotoxinbinding activity of acetylcholine receptor preparations. (A) Detergent extracts: activitiesassayed by ammoniumsulphate precipitation methodY (e e) Foetal acetylcholinereceptor; (o o) adult acetylcholinereceptor. (B) Affinitypurified receptors: activities assayed by using filtration on DEAE cellulose.33 (e e) Foetal acetylcholinereceptor; (o o) adult acetylcholinereceptor.

Polyacrylamide gel electrophoresis of purified acetylcholine receptor SDS-Polyacrylamide gel electrophoresis of purified 125I-labelled foetal and adult human acetylcholine receptor showed, in both cases, four major subunits having Mr 44,000 (-+ 1500; n = 15); Mr 51,000 (-+2000); Mr 58,000 (-+ 1700) and 66,000 (-+2500); the 44,000 subunit being predominant. Varying amounts of a minor polypeptide component with Mr 39,000 and aggregates corresponding to Mr > 100,000 were also seen. Representative radioactivity profiles are shown in Fig. 2. Similar patterns were obtained by silver staining following electrophoresis of unlabelled purified receptor. The presence of 10 mM iodoacetamide throughout the extraction procedure increased the clarity but did not ~,therwise alter the patterns described above. Affinity chromatography of purified foetal or adult receptor on DNase I, either before or after radioiodination, did not change the observed subunit pattern, indicating that actin was not a contaminant. When unlabelled purified receptor was electrophoresed under denaturing conditions and the gels were incubated with 125I-labelled ConA, the major subunit bands with Mr 44,000, 51,000, 58,000 and 66,000 were all radiolabelled. The distribution was, however, different for the two receptor types with the 44,000 band predominantly labelled in the adult receptor compared with the 66,000 band in the foetal receptor (Fig. 3).

Affinity labelling of purified receptor Purified foetal and adult acetylcholine receptor were each labelled with 4-(N-maleimido) [3H]benzyltrimethylammonium and submitted to polyacrylamide gel electrophoresis under de-

G.M. Turnbull et al.

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Isoelectric focusing 125I-labelled purified acetylcholine receptor focused as a single peak at pH 5.1 (-+ 0.2) in the case of the foetal receptor and at pH 5.3 ( + 0 . 7 ) for the adult receptor (Figs 5A and B). Purified acetylcholine receptors labelled with tesI-a-bungarotoxin focused again as single peaks, at pH 5.4 (-+0.4) for the foetal receptor and at pH 5.7 (-+0.3) for the adult form; both peaks were absent when the receptors were incubated with an excess of benzoquinonium chloride (0.01 M) before being labelled with o~-bungarotoxin (Figs 5C and D).

Sucrose-density-gradient centrifugation z-I-a-bungarotoxin labelled purified foetal acetylcholine receptor sedimented on sucrose gradients as a single component with a sedimentation coefficient Se0w= 8.5S (Fig. 6A), whereas

Foetal human acetylcholine receptor

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Fig. 4. Affinity labelling of acetylcholine receptor with 4-(N-maleimido) [3H]benzyltrimethylammonium. Distribution of radioactivity in polyacrylamide gels after electrophoresis under denaturing conditions of affinity labelled (A) foetal and (B) adult purified receptors. Affinity labelling was done as described in Materials and Methods without ( • ) and with ( o ) prior incubation in the presence of c~-bungarotoxin. the similarly labelled adult r e c e p t o r sedimented at S20w= 9.5S (Fig. 6B). B o t h peaks of radioactivity were abolished w h e n acetylcholine r e c e p t o r was incubated with 125I-a-bungarotoxin in the presence of an excess of b e n z o q u i n o n i u m chloride. The difference in S20w values for the two receptor types was confirmed by sedimentation of a mixture of the two on a single gradient when the two peaks r e m a i n e d distinct (Fig. 6C). Results with the foetal r e c e p t o r were o b t a i n e d in three separate experiments.

Inhibition by ConA of binding of a-bungarotoxin to receptor T h e extent to which binding of 125I-labelled a - b u n g a r o t o x i n to purified acetylcholine r e c e p t o r could be inhibited by prior incubation of r e c e p t o r with C o n A was investigated. Increasing concentrations of C o n A led to increased inhibition of labelled a - b u n g a r o t o x i n binding up to a m a x i m u m of 70% inhibition in the cases of both the foetal and the adult receptors. T h e inhibition curves in the two cases were similar (Fig. 7). DISCUSSION T h e a m o u n t s of acetylcholine r e c e p t o r in detergent extracts of foetal h u m a n muscle were consistently 50% higher than those in o u r adult muscle preparations, p r e s u m a b l y reflecting the

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Fig. 5. lsoelectric focusing gels of (A, B) =ZSl-acetylcholincreceptors and (C, D) 1251-e~-bungarotoxin labelled affinity-purifiedacetylcholinc receptors. (A, C) Foetal receptors. (B, D) Adult receptors. Incubation of ~2~I-c~-bungarotoxinwith receptor in the presence of an excess of benzoquinonium chloride completely abolished the peaks of radioactivity in (C) and (D). presence in foetal muscle of extrajunctional receptor. ~j The purified foetal receptor was homogeneous as shown by isoelectric focusing following direct radioiodination and, when subjected to polyacrylamide gel electrophoresis under denaturing conditions, showed four major bands, whether detected as radioactivity in the case of the 125I-labelled receptor, or by silver staining of the unlabelled receptor. The four bands correspond to the (x, [3, ~ and 8 subunits of the electric fish receptor 5 and are all consistently present in both foetal and adult human receptor when purified by the presently described procedure, which is slightly modified, in particular being faster, compared with that previously described. 33 A minor component with Mr 39,000 was occasionally present in both foetal and adult receptors and could well represent a proteolytic degradation product; larger components with M r > 100,000 also occurred and these probably reflect the presence of aggregates of smaller subunits, i 1.25..~2The presence of (x, [3, 3' and ~ type subunits in human acetylcholine receptor confirms earlier suggestions of Lindstrom et al. is made on the basis of interactions of a n t i - T o r p e d o receptor subunit antibodies with unpurified human receptor, and as the number of reported purifications of receptor from vertebrate muscle increases it appears increasingly likely that all such receptors contain four subunits analogous to those of the electric fish, although the exact M,- values and susceptibility to proteases of the indi-

Foetal h u m a n acetylcholine r e c e p t o r

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vidual polypeptides may vary considerably. It also appears probable that the acetylcholine binding site is always carried by the oLsubunit, as demonstrated again here by affinity labelling of both the foetal and adult human receptors. No consistent differences were observed between SDS-polyacrylamide gel patterns of foetal and adult human receptor, whether derived from the ~251-1abelled receptor or from silver staining. When the electrophoresis patterns of unlabelled receptor were incubated with 125I-labelled ConA, however, marked differences were apparent. Thus, although, in both cases, all four major subunits showed bound radioactivity, the 44,000 band of foetal receptor was much less strongly labelled than that of the adult form. Antigenic differences between extrajunctional and .junctional acetylcholine receptors arc well established in the case of denervated and normal adult rat mt"~cle: assay of anti-receptor antibodies in the serum of many patients with myasthenia gravis leads to higher titres when extrajunctional, rather than junctional receptor is used as antigen.143~ Similar differences have also been reported between extrajunctional receptor from embryonic rat muscle and normal adult rat receptor. 3~ Corresponding data from extrajunctional and junctional human acetylcholine receptors are less clear, largely because of problems concerned with the availability and classification of 'denervated" and 'normal' human muscle. Nevertheless, higher anti-receptor antibody titres have been reported for some myasthenic sera when denervated ischaemic muscle rather than normal non-ischaemic muscle is used as a source of acetylcholine receptor, t7,3¢, On the other hand we ourselves compared extrajunctional receptor from foetal human muscle with normal adult receptor in their interactions with myasthenic sera and were tmable to detect significant differences between the binding characteristics of the two receptor types. -~' In further immunological comparisons of extrajunctional and junctional receptors from dencrvated and normal adult rat muscle, Dwyer et al. 7 showed that some myasthenic sera inhibited bmding of ¢x-bungarotoxin to the extrajunctional receptor to a greater extent than to the junctional form. The difference could be reduced but not abolished by treatment of the extrajunctional reccptor with glycosidases, from which the authors concluded that differences between their,junctional and extrajunctional rat receptors werc located closc to the toxin binding site and that carbohydrates contribute significantly to these differences. Our present finding of markedly different distributions of carbohydrate among the receptor subunits of foetal and adult human receptors are relevant to the immunological differences between extrajunctional and junctional rat receptors discussed above, although direct extrapolation from denervated rat to foetal human receptor is clearly not necessarily valid. On our evidence of ConA binding, the most obvious difference between foetal and adult human receptor lies in the lower carbohydrate content of the foetal ¢.,-subunit. Lindstrom et al.l'> have also suggested that structural differences between extrajunctional and junctional rat receptors are at least partially located on the c~-subunits; a conclusion drawn from their obserwltion that antisera to the c~-subunit of T o r p e d o receptor showed a higher titre against receptor from denervated rat muscle than against that of normal adult rats. A relative absence of carbohydrate on the c,-subunit of extrajunctional compared with junctional receptor could explain the presence of extra antigenic sites on denervated rat receptor if carbohydrate served to shield, rather than provide such sites; the effects of glycosidases on denervated rat receptor would be less easy to rationalise. In vicw of the demonstrated lack of ConA binding sites on the ex-subunit of the foetal, compared with adult receptor, we examined the effect of C o n A binding on the binding of c~-bungarotoxin to foetal receptor. No differences were observed between the profiles of ConA-induced inhibition of toxin binding to foetal and to adult human receptors; both attaining a maximum value of 7(1% as concentrations of C o n A were increased. Binding of c,-bungarotoxin to various types of acetylcholine receptor has been shown to be inhibited by C o n A to maximum extents of between 30 and 70% depending on the receptor source. 3"24"2939 The mechanism of inhibition is not clear, but could be explained either in terms of heterogeneity of receptor carbohydrate 3'~ or of random binding of ConA to different receptor sites, occupancy of some of which precludes binding of further ConA, but not toxin, to the toxin-binding site.~ In either case, blockade of toxinbinding could be caused by steric hindrance by ConA bound close to, but not necessarily at the toxin-binding site itself, and need not be affected by different degrees of glycosylation of the ~subunit.

Foetal human acetylcholine receptor

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A c e t y l c h o l i n e r e c e p t o r purified f r o m b o t h f o e t a l a n d a d u l t h u m a n m u s c l e e l e c t r o f o c u s e d as single s h a r p p e a k s w h e t h e r l a b e l l e d by direct i o d i n a t i o n o r by 125I-et-bungarotoxin. In c o n t r a s t to the results of B r o c k e s a n d H a l l , 4 o b t a i n e d with r e c e p t o r s purified f r o m d e n e r v a t e d a n d n o r m a l a d u l t rat m u s c l e , we w e r e u n a b l e to d e m o n s t r a t e significant d i f f e r e n c e s b e t w e e n the isoelectric p o i n t s o f t h e two forms. S u m i k a w a et al. 34 a n d G o t t i et al. 12 similarly failed to d i f f e r e n t i a t e b y isoelectric focusing b e t w e e n r e c e p t o r s purified f r o m d e n e r v a t e d a n d i n n e r v a t e d c h i c k e n 34 a n d r a b b i t 12 muscle. T h e pI v a l u e o f 5.3 f o u n d for le5I-labelled a d u l t h u m a n r e c e p t o r differs m a r k e d l y f r o m t h a t (6.6) p r e v i o u s l y r e p o r t e d by us. 33 H o w e v e r , as p r e v i o u s l y discussed, 33 the l a t t e r v a l u e was o b t a i n e d by using a l a b e l l e d r e c e p t o r p r e p a r a t i o n of v e r y high ~25I c o n t e n t , which c o u l d l e a d to c h a n g e s in c o n f o r m a t i o n a n d isoelectric p o i n t r e l a t i v e to t h o s e of t h e native r e c e p t o r . O u r purified a d u l t a c e t y l c h o l i n e r e c e p t o r p r e p a r a t i o n s s h o w e d '.ittle e v i d e n c e of h e t e r o g e n e i t y , as p r e v i o u s l y r e p o r t e d . This c o n t r a s t s with the results of M o m o i a n d L e n n o n 25 o b t a i n e d with rec e p t o r purified f r o m similar sources. T h e i r p r e p a r a t i o n s h o w e d b r o a d p e a k s in b o t h i s o e l e c t r i c focusing a n d in sucrose d e n s i t y c e n t r i f u g a t i o n ; results which t h e y a t t r i b u t e d e i t h e r to p r o t e o l y s i s o r to m o l e c u l a r h e t e r o g e n e i t y . P r o t e o l y s i s is c e r t a i n l y a m a j o r p r o b l e m in the i s o l a t i o n of acetyic h o l i n e r e c e p t o r f r o m h u m a n tissue 33 a n d o u r purification is, for this r e a s o n , r o u t i n e l y c a r r i e d o u t in the p r e s e n c e of an e x t e n s i v e r a n g e of p r o t e a s e i n h i b i t o r s at 4°C a n d c o m p l e t e d within 27 h. W h e r e a s the purified a d u l t a c e t y l c h o l i n e r e c e p t o r s h o w e d a single s h a r p p e a k with S2ow 9.5 on s u c r o s e d e n s i t y c e n t r i f u g a t i o n , the foetal r e c e p t o r gave rise to a b r o a d e r p e a k with a m a x i m u m at s2~Jw 8.5. This d i f f e r e n c e m a y reflect a r e l a t i v e instability, arising f r o m p r o t e o l y s i s o r s u b u n i t diss o c i a t i o n , o f the purified f o e t a l r e c e p t o r which s h o w e d a r e m a r k a b l y r a p i d loss o f t o x i n - b i n d i n g c a p a c i t y ; a p h e n o m e n o n which, t o g e t h e r with the small a m o u n t s a v a i l a b l e , p r e c l u d e d m e a n i n g f u l a s s e s s m e n t o f its specific activity. T h e significance o f d i f f e r e n t i a l g l y c o s y l a t i o n b e t w e e n the f o e t a l a n d a d u l t forms of h u m a n r e c e p t o r is u n c e r t a i n a l t h o u g h v a r i a t i o n s in a m o u n t a n d d i s t r i b u t i o n o f c a r b o h y d r a t e c o u l d contrib u t e to the k n o w n d i f f e r e n c e s in b e h a v i o u r of the two forms. T h e m e a n s by which a c e t y l c h o l i n e r e c e p t o r s b e c o m e localised at s y n a p s e s a n d c o n c o m i t a n t l y s t a b i l i s e d are not k n o w n b u t it is p o s s i b l e that e x t r a c e l l u l a r m a t r i x m a t e r i a l is i n v o l v e d in these p r o c e s s e s l° a n d that i n t e r a c t i o n of such m a t e r i a l with the m e m b r a n e - b o u n d r e c e p t o r is m e d i a t e d by its c a r b o h y d r a t e c o m p o n e n t . l T h e i d e a t h a t r e c e p t o r d e v e l o p m e n t d e p e n d s u p o n p o s t - t r a n s l a t i o n a l e v e n t s a c c o r d s with similar s u g g e s t i o n s m a d e b y K l a r s f e l d et al. 16 in c o n n e x i o n with t h e i r findings t h a t a single g e n e c o d e s for the a - s u b u n i t in T o r p e d o m a r m o r a t a . Acknowledgements--GMT was in receipt of a post-graduate training award from the Science and Engineering Research

Council. RH and GGL are grateful for support from the Medical Research Council.

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