lb JOWRNU OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemiatry and Molecular Biology, Inc.

Vol. 269, No. 6. Issue of February 11, pp. 4092-4097, 1994 Printed in U.S.A.

Ac~tylcholine Mustard Labels the Binding Site Aspartate in Muscarinic Acetylcholine Receptors*

(Received for publication, August 2, 1993, and in revised form, October 18, 1993)

'hacy A. SpaldingS, Nigel J. M. Birdsall, Carol A. M. Curtis, and Edward C. Hulmeg From the Division of Phvsical Biochemistrv. Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 lAA, unitkd Kingdom

-.

Acetylcholine mustard (AChM) is an analogue of ace- tylcholine (ACh) in which the onium headgroup is re- placed by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat fore- brain have been specifically labeled with [%I]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecu- lar masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation re- veals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [sH]propylbenzilylcholine mus- tard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the d C h R s , Corresponding to Asp-106 (ml sequence), is the site of label attachment.

We have provided strong evidence that the alkylating an- tagonist analogue [3H]propylbenzilylcholine mustard aziridi- nium ([3H]PrBCM)1 labels a conserved aspartic acid residue in transmembrane helix 3 of the muscarinic acetylcholine recep- tors corresponding to Asp-105 in the rat and human m l mAChR sequence (1,2). In the present study, we show that the corresponding agonist analogue, [3Hlacetylcholine mustard az- iridinium ([3H]AChM) labels the same residue as i3H]PrBCM. Although the AChM aziridinium ion has agonist activity, the alkylated receptor is not constitutively activated. A preliminary account of some of these findings has been presented (3).

EXPERIMENTAL PROCEDURES

13H]PrBCM (N-~2,3-~qH~]propyl).N.(2-chloroethyl)-2-~inoethyl ben- zilate, 40 Ci/mmol) and [3HlAChM (N-rnethyl-N-(2-~hloro-l-[~H~lethyl)- 2-aminoethyl acetate, 34.5 Ci/mmol) were obtained by custom tritiation (Amersham Corp.). [3H]AChM was stored in dichloromethane, which was removed by evaporation before the ligand was used. Non-radioactive AChM was synthesized by the method of Jackson and Hirst (41, and its identity was verified by NMR. Its properties were as described (5). The

(United Kingdom) and Merck Sharp and Dohme. The costs of publica- * This work was supported by grants from Medical Research Council

tion 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.

$Recipient of a research studentship from the Medical Research Council and Merck Sharp and Dohme. Present address: Molecular Neu- ropharmacology Section, Dept. of Psychiatry, Medical Alumni Building, University of Vermont, Burlington, VT 05405-0068.

6 To whom corresoondence should be addressed. "el.: 44-81-959-3666: Fax: 44-81-906-4473.

L . - ~~~ ~~

The abbreviations used are: PrBCM, propylbenzilylcholine mus- tard; Ach, acetylcholine; AChM, acetylcholine- mustard; NMS, ( - W -

fast protein liquid Chromatography; CHO, Chinese hamster ovary; DTT, methylscopolamine; mAChR, mmcarinic acetylcholine receptor; FPLC,

dithiothreitol; PAGE, polymacrylamide gel electrophoresis.

mustard was stored as the free amine at -20 "C, and a precipitate of piperazinium dimer, which accumulated slowly (5) , was removed by mi- crocentrifugation before use; the supernatant has retained undimin- ished activity for more than 2 years. The identity of C3HL4ChM with unlabeled AChM was verified by thin layer chromatography in three systems: n-hexane:ethyl acetate (1:l); n-hexane:ethyl acetate (1:8); re- verse phase thin layer chromatography on Whatman KCls in methanol: water:triethylamine (75:25:1).

Unlabeled AChM was cyclized before use by incubation at a concen- tration of 1 m in 20 m NaHEPES, pH 7.4, at 25 "C. The typical yield of aziridinium ion was 70% and was constant between 25 min and 90 min, gradually decaying thereafter with a half-time of >6O min at 25 "C. PHIAChM was cyclized in HEPES buffer or added to reaction mixtures without precyclization, as indicated. r3H1PrBCM was cyclized for 60 min in 10 m sodium phosphate, pH 7.5, at 25 "C before use. Aziridi- nium ion formation by unlabeled mustards was assayed by the addition of 1.5 volumes of an equimolar concentration of sodium thiosulfate, followed by acetic acid to 25 mM, and BDH iodine indicator (British Drug Houses, Dagenham, United Kingdom) to 0.5%. Following incuba- tion at 25 "C for 1 h, the unreacted thiosulfate was back-titrated with 1 rm iodine solution. Blanks, containing no mustard, were analyzed in parallel.

mAChRs were purified from rat forebrain and striatum as described previously (1). Membranes for functional and binding assays were pre- pared from CHO cells transfected with single human mAChR subtypes (6) which were grown to confluence in 30 x 30-cm plates (Nunc) in cr-minimal essential medium enriched with 10% fetal calf serum, 2.4 nm glutamine, 0.07 gfliter penicillin, and 0.12 &iter streptomycin. m e r washing with phosphate-buffered saline, the cells were scraped into phosphate-buffered saline and spun at 700 x g for 10 min. Pellets from 12 plates were homogenized in 50 ml of 20 rn HEPES, 10 m EDTA, pH 7.4, using a glass-in-glass homogenizer, and membranes recovered by centrifugation at 140,000 x g for 30 min at 4 "C. The membranes were washed by homogenization, followed by centri~gation, in the same volume of 20 rm HEPES, 0.1 nm EDTA, and finally in 20 m HEPES, before immediate use in GTPase assays, or freezing and stor- age at -80 "C for binding assays.

Purified mAChRs stored at -80 "C were thawed and gel-filtered on Sephadex G50F equilibrated in 20 m HEPES, 1 m MgClZ, 0.1% digitonin, pH 7.5, to remove carbachol used in the storage buffer. The receptor preparation, typically 10 pmol in 1.8 ml, was added to [3H]AChM, from which the dichloromethane had been removed by evaporation, to give a final radioligand concentration of 1 p. To avoid handling losses and unnecessary dilution of radioligand and receptor, the [3HL4ChM was usually not cyclized before use. AChM cyclizes rap- idly in HEPES-containing buffers, and the aziridinium ion formed is relatively stable; preliminary experiments showed that the efficiency of receptor labeling by [%3%ChM (but not C3HIPrBCM) was hardly af- fected by omission of the precyclization step. To measure nonspecific binding, receptor was treated with lo-* M N-m~thylscopolamine (NMS) before addition of I3H]AChM. Reaction with tSH1PrBCM was carried out in parallel, using 10 ~d cyclized radioligand. Incubation was carried out at 15 "C for 16 h.

Following addition of 0.1% SDS and concentration to 100 pl(3000 X

g, 4 "C, Centricon 10,Amicon), the reaction mixture was fractionated on a Superose 12 column (Pharmacia LKB Biotechnology Inc.) in 50 nud Tris-Cl, 0.1% SDS, pH 7,.8 as described previously (1, 2). The peak fractions were pooled. The incorporation of L3H1PrBCM was -4 nmoVmg protein (assessed by measurement of absorbance at 214 nm), and that of [3HIAChM was 0.5-0.75 nmoVmg protein. Hydroxylamine treatment of labeled mAChRs, carboxymethylation with and without prior reduction with Dm, detergent exchange, CNBr cleavage, and

4092

6 1 L 4 6 1 ?+F

Muscarinic Agonist Binding

log [drug1

AChM measured by GTPase assay. The GTPase response of ml, m2, FIG. 1. Representative dose-response curves for ACh and

and m4 mAChRs to ACh and AChM was measured using 30 nCi (10 m) [y3*P]GTP as substrate (see "Experimental Procedures"). Membranes were prepared from CHO cells expressing ml (a) , m2 (b) , and m4 (c) mAChRs. ml and m4 membranes were freshly prepared, but m2 mem- branes had been stored at -80 "C before use. Drug concentrations for AChM refer to aziridinium concentration. Pi release values represent mean t: range for duplicate assays, except for basal values where n = 6. 0, ACh; 0, AChM; At?, lo4 M ACh or AChM + lo4 M atropine; Basal, water added in place of agonist. Solid lines represent the best fit to the Hill equation. Parameter values (-log ECSO (ns)) were as follows: a, ml: ACh 5.6 (0.5), AChM 5.4 (0.5); b, m2: ACh 6.6 (0.61, AChM 6.5 (0.3); e, m 4 ACh 6.9 (0.61, AChM 6.3 (0.7). Maximal stimulation by ACh (as % of basal) was 186 f 10 (ml; n = 5), 150 t: 12 (m2; n = 21, and 190 * 10 (m4; n = 6). Mean stimulation by AChM was 64% (ml) and 100% (m2, m4) of the stimulation given by ACh.

SDS-PAGE of intact and digested receptors were essentially as de- scribed (1, 2). For sequencing, reaction mixtures containing CNBr- cleaved radiolabeled mAChRs were applied to Polybrene-coated glass fiber discs, and dried, prior to automated Edman degradation in an Applied Biosystems Inc. model 477A pulsed-liquid phase proteid peptide sequenator with operational modificatio~ as described (2). The tritium radioactivity released in each sequenator cycle was measured.

The binding of (-~-N-~3Hlmethyl~opolamine ff3H1NMS, 85 Cilmmof; DuPont NEN) to mAChRs in membranes from transfected CHO cells typically employed a receptor concentration of 50 PM, and was assayed following separation of bound from free ligand by filtration using a Brandel cell harvester onto Whatman GFB glass fiber paper presoaked in polyethylene imine (0.1%). Filters were rapidly (.e20 s) washed twice with 5 ml of water and dried for 2 min in a microwave oven. Bound ligand was measured by liquid scintillation counting. Binding to puri- fied receptors in solution was assayed by gel filtration of 0.2-ml aliquots on 2-ml columns of Sephadex G50 (medium or fine, depending on the aims of the experiment) equilibrated at 4 "C in 20 n m HEPES, 0.1% digitonin, pH 7.5. Bound E3H1NMS was recovered in the first 1 ml of eluate and measured by liquid scintillation counting.

GTPase activity stimulated by muscarinic agonists in the mem- branes of transfected CHO cells was measured by an adaptation of the methods of Cassel and Selinger (71, and Hilf and Jakobs (8). Membranes were mixed with agonist and 30 nCi 1-10 m) [Y-~~PIGTP (specific activity 30 CUmmol, Amersham Corp.) in 100 pl of 20 mM HEPES, 100 m NaCl, 5 mM MgC12, 0.1 m EDTA, 3 m ATF? Incubation was for 15 min at 30 "C. The reaction was stopped by cooling on ice and adding 500 pl of acidified powdered charcoal (5% charcoal (Sigma C5610), 0.1% bovine serum albumin, 20 n m phosphoric acid). The mixture was left for 10 min, the charcoal pelleted, and the 32Pi contained in a 400-pl sample of the supernatant measured by scintillation counting.

RESULTS

AChM Aziridinium Activates Muscarinic Receptors-The agonist activity of AChM aziridinium was assessed by stimu- lation of the GTFase activity of G-proteins coupled to mAChRs

site 4093

I z

0 20 40 60 time (min)

FIG. 2. Blockade of membrane-bound and purified, soluble mAChRs by AChM. a, alkylation of m l mAChRs in membranes from CHO cells expressing m l mAChRs. 28 p cyclized AChM was added to freshly prepared membranes in GTPase assay buffer (see "Experimen- tal Procedures"). Aliquots (0.1 ml) of this mixture were incubated for 2-60 min at 30 "C. Alkylation was stopped by the addition of 2 ml of 2 m r3HINMS and 2 m sodium thiosulfate. The tubes were incubated for 2 h at 30 "C to allow L3HINMS to replace reversibly bound AChM before assay by rapid filtration (see "Experimental Procedures"). The curve shows a fit to a single exponential, with a first-order rate constant of 1.15 x s-l. NS represents [3HINMS binding in the presence of atropine. b, AChM inhibition of [3HlNMS M) binding (0) and irre- versible blockade of binding sites (0) in a purified mAChR preparation from rat forebrain. Inhibition was measured by incubation of mAChRs (0.13 pmollml) with I3H1NMS (lov9 M) and graded concentrations of cyclized AChM in 20 m HEPES, 0.1% digitonin, pH 7.5, at 15 "C for 90 min before assay of bound r3H1NMS by gel filtration on G50M columns (see "Experimental Procedures"). Alkylation was measured by preincu- bating mAChRs (1.2 pmol/ml, 250 pl) with graded concentrations of cyclized AChM for 90 min at 15 "C. mAChRs were separated from free AChM by gel filtration of 2 0 0 4 aliquots on 2-ml columns of G50F in HEPES/digitonin at 4 "C. Receptors eluted between 0.6 and 1.1 ml. Residual binding capacity in the eluate was measured by the addition of a receptor-saturating concentration M; 30 x &) of PHINMS fol- lowed by incubation for another 60 min at 15 "C, and assay by gel filtration (see "Experimental Procedures"). Acetylcholine gave no inhi- bition of binding at concentrations of up to M using this protocol. Points are the mean A range of duplicate measurements from a repre- sentative experiment, which was repeated four times with the same results.

in the membranes of CHO cells transfected with single mAChR subtypes (9). Fig. 1 shows that ACh stimulated this activity by about Mold at ml , m2, and m4 mAChRs; m3 mAChRs gave a very low signal with this assay and were not studied further. AChM aziridinium stimulated GTPase activity, with ECso val- ues similar to those given by ACh itself (see legend to Fig. 1). At m2 and m4 receptors, the E,,, values were also comparable for ACh and AChM. At m l receptors, the irreversible agonist gave a lower E,,,. As shown below, this probably results from al- kylation of the m l mAChRs during the course of the experi- ment; alkylation by PrBCM gives a similar reduction of E,ax.2

AChM Aziridinium Blocks nAChRs in Membran.es and in Purified Preparations from Rat Forebrain-A near-maximally effective concentration (28 p) of AChM aziridinium alkylated up to 90% of the receptors in a membrane fraction from CHO cells transfected with m l mAChRs, with a half-time of 10 min at 30 "C, corresponding to a n alkylation rate constant of 1.2 x

s-l (Fig. 2a); the latter value agrees well with the rate of blockade of mAChRs in guinea pig ileum longitudinal muscle (6) and is -3-fold slower than the corresponding rate constant for PrBCM.3 AChM alkylation of purified mAChRs from rat forebrain was conducted at 15 "C in digitonin solution, condi- tions found previously (1) to give a reasonable compromise between receptor stability ( ~ 2 0 % loss in activity in 90 min) and

S. Lazareno, personal communication. T. A. Spalding, unpublished results.

4094

****

Muscarinic Agonist Binding Site

100 1

25 30 35 40 45 50

Fraction Number 200 97 69 46 30 22 14 kDa - _ " - "

100 h A U

0 I

0 20 40 60 80 100 R F %

FIG. 3. Labeling of purified mAChRs from rat forebrain with [SHIAChM. a, mAChRs (6 pmoYml) labeled with r3H1AChh4 (1 p a ) in the absence or presence of NMS M) as described under "Experi- mental Procedures" were concentrated, then fractionated by gel filtra- tion on a Superose 12 column in 50 m~ Tris-Cl, 0.1% SDS, pH 7.8. 0.25-ml fractions were collected and samples taken for liquid scintilla- tion counting. The solid line shows the profile given by 8.6 pmol of receptor labeled in the absence of NMS; the broken line shows the profile given by receptor labeled in the presence of NMS. Overall recov- ery of NMS-sensitive F'HIAChM binding sites was 22% of the total receptor applied. Fractions marked with an asterisk were pooled. b, SDS-PAGE of reduced, carboxymethylated [3HlACh"labeled mAChRs (3000 dpm) pooled from a. Overall recovery of radioactivity was -70%, 40% of which was under the peak of -69 kDa, and 20% in the region above 200 kDa. c, SDS-PAGE of reduced, carboxymethylated [3HlPrBCM-labeled mAChRs (5000 dpm). Overall recovery of radioac- tivity was -53%, of which 60% was under the peak of -69 kDa.

alkylation rate (reduced approximately 3-fold compared to 30 "C). Under these conditions, AChM aziridinium inhibited the binding of the reversible antagonist [3H]NMS M) and blocked up to 85% of free mAChR binding sites in 90 min (Fig. 2b; Ref. 10). The IC60 values for inhibition (-log ICso = 4.8 f 0.05; n = 3) and block (-log IC50 = 4.86 2 0.25; n = 4) were in the same range as the concentrations needed to activate GTPase activity (Fig. l a ) .

PHlAChM Specifically Labels the mAChR Polypeptide in a Purified Preparation from Rat Forebrain-Purified rat fore- brain mAChRs were labeled with [3H]AChM using a protocol similar to that developed for labeling with [3H]PrBCM (1, 2). Because of its lower affinity for the receptors, the concentration of f3H1AChM employed was higher (1 p ~ ) than for L3H1PrBCM (10-30 nM), although still not receptor-saturating and the in- cubation time was longer than used routinely for L3H]PrBCM labeling (1, 2). AChM cyclizes readily in HEPES-containing buffers (see "Experimental Procedures"); therefore, to avoid handling losses, the radioligand was added without precycliza- tion to purified receptor, and the reaction was allowed to pro-

1 2 3 Time (days)

FIG. 4. Release of [sHIACbM from [SHIACh"labeled mAChRa by hydroxylamine. 20-pl aliquots of [3HlAChh4-labeled mAChRs in 50 m~ Tris-C1, 0.2% SDS were treated with an equal volume of fresh 2 M hydroxylamine (0) or 2 M NaCl (0) at 22 "C. One volume of fresh 2 M hydroxylamine (or NaCl) was added daily, to compensate for any hy- drolysis. Label cleaved from the receptor by the action of hydroxylamine was assayed by gel filtration as described (1).

ceed for 16 h at 15 "C (see "Experimental Procedures"). Calcu- lation shows that protection of 99% of the binding sites in a prolonged incubation with a high concentration of site-directed affinity label with the same alkylation rate constant as AChM requires a competitor concentration greater than lo4 times its &value. M NMS (Kd 3 x 10-lo M; Ref. 11) was employed for this purpose. Following concentration (see "Experimental Pro- cedures"), the reaction mixtures were purified further by FPLC gel filtration on a Superose 12 column in a buffer containing 0.1% SDS as described (1, 2). Fig. 3a shows that [3HlAChM radioactivity peaked in fractions known to contain mAChRs (1, 2); mAChRs labeled with L3H1PrBCM eluted in the same posi- tion (not shown). The incorporation of radioactivity into these peak fractions was significantly suppressed when labeling was performed in the presence of NMS. Some radioactivity eluted in the void volume of the column; this was not NMS-sensitive. Typically, 20-30% of the receptor binding sites present at the start of the alkylation reaction were recovered in L3H1AChM- labeled form in fractions 3 6 4 2 (Fig. 3a). This value is lower than found for [3H]PrBCM (-60%; Refs. 1 and 2), probably reflecting the lower alkylation rate constant and less favorable occupancy of the receptor during the reaction. The stoichiome- try of labeling was -0.75 nmol/mg protein; again, this was lower than the corresponding value obtained for i3H1PrBCM (3 nmol/mg protein) in a parallel labeling reaction.

SDS-PAGE of individual fractions showed a single labeled species of molecular mass 60-70 kDa (not shown). The peak fractions (fractions 37-41) were pooled, reduced, and carboxy- methylated. SDS-PAGE showed a major radiolabeled peak of molecular mass -67 kDa (Fig. 3b) identical in its electropho- retic behavior to [3HlPrBCM-labeled mAChRs (Fig. 3c). A smaller amount of radioactivity was present in species of over 200 kDa, which may have contained receptor aggregates.

The PHIAChM-Receptor Bond Is Hydroxylamine-sensitive- The [3H]AChM-receptor bond was cleaved by incubation with a 1 M concentration of the strong nucleophile hydroxylamine, under denaturing conditions (Fig. 4). Over 60% of the label was removed in 24 h at room temperature. The AChM-receptor bond appears more readily cleaved than the [3HlPrBCM-recep- tor link; there was substantially faster release of L3H1AChM than [3H]PrBCM in control incubations (Ref. 1; data not shown).

Cyanogen Bromide Cleavage of PHIAChM-labeled mAChRs -Fig. 5 shows that CNBr cleavage of fully reduced carboxy- methylated [3H]AChM-labeled receptors yielded a peptide map with the same qualitative features found previously for

Muscarinic Agonist Binding Site 4095

[3H~PrBCM-la~led mAChRs (2). In particular, two small pep- tides of -2.4 and -3.9 kDa were evident, as was a minor product of -29 Mla. There was also some apparently uncleaved material. In all comparative peptide mapping experiments, the r3H]AChM and [3HlPrBCM-labeled mAChRs have given iden- tical distributions of peptide molecular masses. These included digestions with Glu-C (15 pdml), which gave a major product of 40 kDa and a minor product of 16 kDa (data not shown), as reported by Uchiyama et al. (12) for [3HlPrBCM-labeled mAChRs purified from porcine brain. The glycosylation state of

46 30 22 14 6.5 3.412.4 kDa zooo-, - - . - - -

0- a 20 40 60 80 100

RF %

FIG. 5. Cyanogen brodde cleavage of t ~ ~ l A ~ - l a ~ l ~ mAC- analysis by SDS-PAGE. 0.8 pmol(17,OOO dpm) of redueed, ~ r ~ ~ e t h y l a ~ [ 3 H ~ C ~ - l a b e l e d mAChRs was cleaved with CNBr (20 h; see "Experimental Procedures"), After neutralization with NaOH, the reaction mixture was analyzed by SDS-PAGE. Overall re- covery of r a ~ o a c t i v i ~ was 60%. The m o l ~ ~ l a r size standards used were Bio-Rad "Rainbow" standards. in this experiment, the 3.5- and 2.35-kDa standards were not separated. However, in subsequent ex- periments, it was possible to assign approximate molecular masses of 3.9 and 2.4 kDa to the CNBr cleavage products of lowest molecular mass.

200 97 69 46 30 22 14 6.5 3.4 2.4

\>ff 1 1 1 1If 3007

i (a'

the 2.4- and 3.9-kDa CNBr peptides was not addressed directly, but it should be noted that the apparent molecular mass of the carboh~drate associated with the mAChRs and any glyco- sylated digestion products identified so far is -15 kDa (1, 2, 12).

The 2.4- and 3.9-kDa ~H]ACh~-labeled Peptides Contain a ~ i ~ u ~ ~ ~ - ~ o ~ d e d Cys Residue-Exhaustive CNBr cleavage of 13H]AChM or [3H]PrBCM-labeled mAChRs in which free sulf- hydryl groups had been alkylated without prior reduction of endogenous disulfide bonds gave labeled peptides whose appar- ent molecular masses were mainly in excess of 6.5 kDa, and only a minor yield ofsmaller cleavage products (Fig. 6, a and e). On reduction of the cleavage mixture with Dm, fotlowed by carboxymethylation of newly liberated sulfhydryl groups, the larger products disappeared to be replaced almost completely by peptides of -2.4 and -3.9 ma, with a small fraction of the -29-M)a peptide (Fig. 6, b and d 1. It should be noted that the relative yield of these peptides partly depends on the complete- ness of the cleavage (2).

This result is compatible with the presence of a Cys residue within the low molecular mass labeled peptides, which, on CNBr digestion without reduction, mostly retained a disulfide linkage to peptides derived from cleavages elsewhere in the sequence. This agrees with our report that the 2.4- and 3.9-kDa [3HlPrBCM-labeled peptides contain a Cys residue that can be alkylated with ~-(3H]ethyImale~mide after reduction with DTT (2). The generation of a small amount of the low molecular mass peptides without DTT treatment may have arisen from limited scission of the disulfide bond as a result of thioi~disul- fide interchange, and the presence of more than one product may reflect the presence of multiple mAChR subtypes in the purified preparation (1,2), as well as incomplete cleavage.

Edman ~ e g r ~ d a t i o ~ of CNBr-cleaved ~ H l A ~ h ~ - l a ~ l e d

X X , 97 69 4630 22 14 6.5 3.4 2.4

\Vf I 1 1 L 11 Jl

200

0 20 40 60 80 100 0 20 40 60 80 1 0 0 0

f?F ?Q

FPLC-purified PHIAChM and [SH]PrBCM-labeled mAChRs were concentrated 10-fold to 500 m d Tris-Cl,l% SDS, pH 7.8, and carboxymethylated Fro. 6. CNBr cleavage of non-reduced [ $ ~ A C ~ and [ ~ H l ~ B C M - ~ ~ l ~ mAChRe: effect of subsequent reduction with D".

with 2 m~ iodoacetic acid for 22 h a t 4 "C to block free SH groups. The reaction mixtures were gel-filtered on G50F m 0.01% SDS to remove excess

volume of Nzpurged 50 m~ HCl, containing 60 m&mI CNBr, and 100 p&mI tryptophan, and allowed to react at 22 "C for 67 h in the dark, under salts as described (2). The eluates, containing 20,000 dpm of labeled protein, were concentrated 20-fold, to 100 pl, then supplemented with an equal

N2. The cleavage mixtures were gel-filtered on Sephadex G25F in 50 m~ M5Cf,O.l% SDS, pH 7.8. During this step, 5% of the [3HlAChM label was recovered as hydrolyzed mustard. Following a 10-fold concentration, to 500 m~ Tris-C1, 1% SDS, half of each reaction mixture was reduced with 10 m~ D m (37 "C, 2h, NZ). The samples were further carboxymethylated with 22 n m iodoacetic acid. Excess buffer and reagents were again removed by gel fikration on Sephadex G25 F in Laemmli b a e r diluted 1:lO. Following a 10-fold ~ n ~ n t r a t i o n , the samples were analyzed by SDSPAGE. a, CNBr-cleaved F'HIAChM mAChRs (non-reduced); b, CNBr-cleaved r ~ ~ ~ C ~ mAChRs (reduced); c, CNBr-cleaved r3HIPrBCM mAChRs (non-reduced); d, CNBr-cleaved [SHIPrBCM mACh% (reduced). Overall recoveries of radioactivity were 2430% for the PHIAChM- labeled samples and 3040% for the [3HlPrBCM-labeled samples, resecting cumulative losses and the greater lability of the [SHIAChM-receptor bond.

4096 Muscarinic Agonist Binding Site

”1

0 ‘ I I I I I 1 1 , 5 10 15 20 25 30 35 40

Cycle Number

[sHIAChM and IBHIPrBCM-labeled mAChRs. r3H1AChM t 110,000 FIG. 7. Edman degradation of CNBr cleavage products of

dpm)- and f3H1PrBCM (130,000 dpm)-labeled mAChRs were reduced, carboxymethylated, cleaved with CNBr, applied to a Polybrene-coated glass fiber disc and subjected to 40 cycles of Edman degradation as

the radioactivity of the thiazolinone amino acid derivatives recovered described under “Experimental Procedures.” For each sequenator cycle,

was measured. a, [ 3 H l A C ~ - m A C ~ ; b, [3HlPrBCM-mACh~.

mAChRs-Edman degradation of labeled CNBr cleavage prod- ucts can be used to provide more detailed information about the exact position of attachment of a radiolabel within a peptide sequence (2). Edman degradation of CNBr-digested t3H]AChM- labeled mAChRs gave release of radioactivity commencing at sequenator cycle 26 (Fig. 7u). An identical result was obtained from [3H1PrBCM-labeled receptors (Fig. 7b), in complete agree- ment with previous results in which Edman degradation was applied following CNBr cleavage of peptides that had been isolated after a preliminary Lys-C proteolysis step (2). The peak shapes were consistent with a repetitive yield of 93%, which is compatible with the efficiency of the Applied Biosystems se- quenator operating at the picomole level.

These results provide further evidence that E3H]AChM and f3H1PrBCM modify the same residue in the receptor sequence, and suggest a site of label attachment 26 amino acids C-termi- nal to a methionine residue. The yield of radioactivity in these sequencing experiments was less (-2.5%) than found in our previous study (-6%; Ref. 2). This was probably because CNBr cleavage of the intact mAChR polypeptide chain used in the present experiments to avert cumulative handling losses was somewhat less efficient than the cleavage of isolated N-termi- nal proteolytic fragments used in our previous study, at least on the scale necessary for sequencing experiments, leading to a reduced yield of sequenceable N termini. In the case of the PHIAChM receptor, the relative lability of the ligand-receptor bond (Fig. 4) may have contributed to washout of radioactivity during the first sequenator cycle and to reductions in yield in later cycles. These factors are additional to side reactions lead- ing to N-block of peptides, which can occur prior to and during sequencing (2 , 13).

GTPase GTPase 3H-NMS NMS ACh capacity

FIG. 8. AChM-labeled mAChRe are not trapped in an active state. Freshly prepared membranes from CHO cells transfected with ml or m4 mAChRs were resuspended in 20 m~ HEPES, 100 m~ NaCl,

with 1 p~ ACh (Q), 1 p~ AChM ( ), or buffer (m) for 15 h at 20 ‘C. A 5 m~ MgCl,, 0.1% EDTA, and divided into aliquots, which were treated

PHINMS (2 n ~ ) binding assay was carried out on randomly selected aliquots of m l or m4 mAChRs, which were diluted 1:20 and incubated for 2 h at 30 “C to allow the f3H1NMS to replace any reversibly bound ligand. Nonspecific binding was defined with 1 p~ atropine. b, m l mAChRa (n = 4); d , m4 mAChRa (n = 4). 170 JIM (ml) or 1 m~ (m4) unlabeled NMS was added to half of the remaining tubes and allowed to equilibrate for 3 h to replace reversibly bound ligand. 1 m~ ACh was added to the remaining tubes. A GTPase assay was then cam& out as described (see “Experimental Procedures”). a, ml mAChRs f n = 4); e, m4 mAChRs (n = 8). The experiment was replicated three times, with the same results.

AChM-alkylated d C h R s Are Not Permanently Acti- uated-It is evident that alkylation of forebrain mAChRs by both AChM and PrBCM is highly specific. An important issue is whether the receptor is trapped in an active state after irre- versible reaction with the agonist analogue. To answer this question, membranes from CHO cells transfected with m l and m4 mAChRs were exposed to 1 AChM for 16 h at 20 C. This led to blockade 40% or more of the total mAChR binding sites, assayed with a receptor-saturating concentration of t3H1NMS (Fig. 8, b and d 1. The alkylated membranes were either treated with an excess of unlabeled NMS to displace noncovalently bound AChM, or with ACh to assay the residual agonist-stimu- lated GTPase activity. Controls were exposed to ACh (1 p) or buffer instead of AChM before further treatment with NMS or ACh as above. GTPase activity was assayed as described (see “Experimental Procedures”; Fig. 1).

The AC~-alkylated mAChRs showed no evidence of activity in the presence of NMS, as would have been anticipated had they been trapped in an active state (Fig. 8, a and c). Instead, the alkylated membranes showed ACh-stimulated activity, which was 75% (ml) to 85% (m4) that of the ACh-treated con- trols. This suggests that the non-alkylated receptors retained activity, and is consistent with the presence of a degree of receptor reserve for the GTPase response (9).

DISCUSSION AChM aziridinium is a close analog of the parent molecule,

ACh, and retains the ability to act as an agonist at muscarinic receptors. The chemically reactive headgroup of AChM is an effbctive alkylating agent for both membrane-~und and soluble mAChRs. The reaction is sufficiently specific to enable mAChRs purified from rat forebrain to be labeled in an NMS- sensitive manner by [3HIAChM. The properties of the [3H]AChM-mAChR link are parallel to those of the link formed by the alkylating antagonist analog, I3H1PrBCM. The label is cleaved from the protein by hydroxylamine, is found within

Muscarinic Agonist Binding Site 4097

CNBr peptides of molecular mass 2.43.9 kDa, is close to a disulfide-bonded Cys residue, and a site of labeling is localized 26 residues C-terminal to a CNBr cleavage site. This supports the proposal that the conserved Asp in TM3 of the mAChRs, corresponding to Asp105 in the rml mAChR, is the major site of esterification by AChM, as for PrBCM (1,2) and that the site of CNBr cleavage is the Met conserved in all the mAChR sub- types corresponding to Met-79 (rat m l sequence), although we cannot exclude the possibility of lower levels of labeling at other positions. As in our previous study (21, we have not observed release of radioactivity after 17 sequenator cycles, which would arise from cleavage at Met-88 (rml sequence), suggesting that very short mustard-labeled peptides may immobilize or se- quence poorly; electrophoretic analysis has shown that the 2.4- kDa [3HlPrBCM-labeled CNBr peptide is retained poorly rela- tive to the 3.9-kDa peptide on amino-derivatized glass beads (2).

A difference between the reagents is that the alkylation rate constant of AChM i s about 30% of that of PrBCM, possibly indicating more efficient stabilization of the transition state of the latter than of the former reaction. This, in combination with the much lower affinity of the agonist mustard analog, leads to lower yields of [3HJAChM-alkylated receptor than are obtained with [3H]PrBCM. We cannot entirely exclude the possibility that [ 3 H ] A C ~ ~fferentially alkylated a subpop~ation of the mAChRs in the purified preparation, although there is no evi- dence of subtype selectivity with this reagent.

AChM aziridinium ion can activate mAChRs. This does not imply that the receptor is in an active conformation in the transition state of the alkylation reaction. Certainly there is no evidence of activity after alkylation. This agrees with the ma- jority of studies on the agonist activity of AChM (5) and its N-ethyl analogue (14,15), although there has been a suggestion that acetylethylcholine mustard aziridinium may be able to exert an irreversible inhibitory effect on potassium-stimulated ACh release from synaptosomes (16). We know from the clas- sical pharmacology of the mAChRs that the stereochemistry around the headgroup of ACh is critical for agonist eEcacy (reviewed in Ref. 17), and it is clear that the geometry of an ester linkage must be very different from that of an optimum

onium-carboxylate ionic bond. Site-directed mutagenesis stud- ies on the AChM-labeled Asp suggest that the interaction be- tween the agonist headgroup and carboxylate side chain is critical for attainment of the activated, G-protein-coupled state of the receptor, but is less i m p o ~ n t for agonist docking (181.* It appears that the aziridinium headgroup of AChM can make this critical interaction, but that the precise positioning of the headgroup within the receptor structure is disrupted by forma- tion of the ester link during the subsequent reaction.

Acknowledgments-We are indebted to Alastair Aitken and Alan Harris for peptide sequencing and to John Come for synthesis of ace- tylcholine mustard and for commenting on the manuscript.

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Iished data. * K. M. Page, P. G. Jones, C. A. M. Curtis, and E. C. Hulme, unpub-