©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of Recombinant Porcine m2 Muscarinic Acetylcholine Receptor from Chinese Hamster Ovary Cells
CIRCULAR DICHROISM SPECTRA AND LIGAND BINDING PROPERTIES (*)

(Received for publication, January 9, 1995; and in revised form, May 22, 1995)

Gary L. Peterson (1) Arazdordi Toumadje (1) W. Curtis Johnson , Jr. (1) Michael I. Schimerlik (1) (2)(§)

From the  (1)Department of Biochemistry and Biophysics and the (2)Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The recombinant porcine m2 muscarinic acetylcholine receptor (rPm2R) from Chinese hamster ovary cells has been purified to homogeneity. Two mg of purified rPm2R, with a specific activity of 12 nmol of R-(-)-quinuclidinyl benzilate/mg of protein, were obtained from 30 ml of packed Chinese hamster ovary cells. The apparent molecular mass (78.5 kDa) and specific activity for the rPm2R preparation were the same as that for the Pm2R purified from atrial tissue, but the yield was 100 times greater. Purified rPm2R bound agonist and antagonist with the same affinities and coupled to the inhibitory guanine nucleotide-binding protein with the same efficiency as the purified native atrial Pm2R. Ligand binding studies were consistant with a single class of antagonist binding sites but two subclasses of agonist binding sites. The fraction of rPm2R having high affinity for agonists was increased by mM Mg, low detergent concentration, and low temperature. Circular dichroism spectra obtained for the purified rPm2R with and without agonists were indistinguishable, but spectra for the antagonist-occupied receptor showed reproducibly deeper characteristic negative deflections at 208 and 220 nm. Secondary structure analysis of the CD spectra predicted 53% -helix for the free receptor and 49% -helix for the R-(-)-quinuclidinyl benzilate-receptor complex.


INTRODUCTION

mAcChRs()belong to the family of seven-transmembrane domain receptors that transduce extracellular neurotransmitter signals by activating G proteins within the cell. Effector systems activated by muscarinic agonists include inhibition of adenylyl cyclase and stimulation of phosphatidylinositol hydrolysis (1) . Five mAcChR subtypes have been identified, and most tissues that have muscarinic activity express multiple subtypes(2, 3, 4) . Studies of purified receptor alone and in reconstitution with other purified signaling components permit analysis under more defined and tractable conditions. However, the supply of purified mAcChR like that of other G protein-coupled receptors is limited.

The mAcChR was purified from heart tissue with a yield of 30 µg of protein (5) and from brain tissue at yields of 6-18 µg(6, 7) . These yields are similar to those for other G protein-coupled receptors from natural sources(8, 9, 10, 11, 12, 13) . Natural sources may also contain multiple subtypes that may be difficult to resolve. Recombinant human -adrenergic receptor and Hm1 and Hm2 muscarinic receptors were purified from baculovirus-infected insect Sf9 cells(14) , with yields for the rHm2R estimated at 6 mg of protein/liter of culture with a specific activity of 2 nmol/mg (10-20% active), and at 40 µg of protein/liter of culture at a specific activity of 8 nmol/mg.

In this paper, we report the purification of fully active recombinant Pm2R (12 nmol/mg) from a line of overexpressing CHO cells (15) with yields in the mg range, up to 100-fold greater than that achieved for the atrial Pm2R(5) . Sufficient material was obtained to complete ultraviolet circular dichroism studies analyzing protein secondary structure in the presence and absence of ligands.

Agonists interact with the purified atrial mAcChR(5) , purified brain mAcChRs (16, 17) and purified brain A adenosine receptors (13) at two subclasses of sites. The assay conditions affecting the equilibrium binding parameters and the distribution between high and low affinity states were examined for the interaction of carbachol with the purified rPm2R.


EXPERIMENTAL PROCEDURES

Materials

WGA was purified according to Kahane et al.(18) and coupled to Affi-Gel 10 (Bio-Rad) at 20 mg of WGA/ml of resin. ABT prepared according to Haga and Haga (19) was coupled to agarose at 1 µmol/ml resin. Chitotriose was prepared according to Rupley(20) . Cholate, recrystallized three times as the free acid, R-(-)-hyoscyamine, acetylcholine, and gallamine were from Sigma. Carbachol and (+)-pilocarpine were from Aldrich. QNB and oxotremorine M were from Research Biochemicals International. [H]QNB (30-46 Ci/mmol), [S]GTPS (1000-1500 Ci/mmol), and [-P]GTP (30-44 Ci/mmol) were from DuPont NEN. Protease inhibitors, purchased from either Sigma or Boehinger Mannheim, were prepared as 1000-fold concentrated stocks: 1 mg/ml pepstatin A and 17 mg/ml phenylmethylsulfonyl fluoride in dry methanol; 1 M benzamidine, 10 mg/ml bacitracin in 95% ethanol; 2.5 mg/ml leupeptin, 2.5 mg/ml aprotinin, 1 mg/ml E-64 in HO. Cell growth media was FD (1:1 mix of Ham's F-12 medium:Dulbecco's modified Eagle's medium) plus 10% calf serum.

Cell Culture

A transfected clone of dhfr CHO cells expressing high levels of rPm2R (15) was adapted to spinner culture and grown in 4-liter spinner flasks containing 1.5 liters of growth media. Expression levels were increased 5-9-fold by treatment with 5 mM sodium butyrate for 15-18 h prior to harvesting. Butyrate treatment can increase gene transcription in CHO cells(21) , which may be due to hyperacylation of histones(22) . Cells were harvested by centrifugation at 4000 rpm for 5 min at 4 °C. Cells from each spinner flask were washed with 45 ml of CHM (250 mM sucrose, 50 mM EDTA, 1 mM EGTA, 25 mM imidazole, pH 7.4) plus protease inhibitors by centrifugation at 1500 g for 2 min in a clinical centrifuge, resuspended in 10 ml of CHM plus protease inhibitors and frozen at -80 °C.

Receptor Purification

Atrial Pm2R was purified as described previously(5) . Purification of the recombinant Pm2R was significantly modified from that for the atrial preparation. Protease inhibitors were added to frozen CHO cells during thawing and to all solutions before use. 30-35-ml batches of packed cells expressing 1-2 10 surface receptors/cell were homogenized under argon for 2 20 s with a PTA 10S polytron probe operated at 60% of maximum speed. The homogenate was centrifuged at 1500 g for 1 min in a clinical centrifuge, brought to 120 ml with CHM, and layered over sucrose gradients prepared in Beckman SW28 rotor tubes. Each tube contained 9 ml of 42.5% sucrose and 9 ml of 20% sucrose in 5EI buffer (5 mM EDTA, 1 mM EGTA, 25 mM imidazole, pH 7.4) and 20 ml of homogenate. After centrifugation at 27,000 rpm for 1 h at 4 °C, the membrane fraction was collected from the 20-42.5% sucrose interface. Homogenization and sucrose gradient steps were repeated on the pellet, and the combined membrane fractions were diluted with EI buffer (1 mM EDTA, 25 mM imidazole, pH 7.4) and pelleted by centrifugation at 70,000 g for 1 h at 4 °C. The pellet was resuspended to 20 ml in EI buffer and assayed for total protein by the Lowry Folin phenol method(23) .

Membranes were adjusted to 4.5 mg of protein/ml with EI buffer, and the rPm2R was solubilized with digitonin/cholate (24) by adding ¼ volume of 10 D/C-EI buffer. After 10 min. at room temperature, the mixture was centrifuged at 70,000 g for 1 h at 4 °C. The supernatant was saved on ice, and the pellet was resuspended in EI buffer with a glass Teflon homogenizer to a total volume equal to one-half of the initial volume and extracted again with ¼ volume of 10 D/C-EI buffer.

Combined detergent extracts were diluted with an equal volume of EI buffer, brought to 5 mM MgCl, combined with 40-50 ml of freshly recycled WGA-agarose, and mixed overnight at 4 °C. WGA agarose, stored in 0.2 M N-acetylglucosamine, was recycled with 130 ml of 0.1 M acetic acid, 250 ml of EI buffer, and finally 50 ml of 5 mM MgCl in D/C-EI buffer. The next day the mixture was transferred to a 3.8 16-cm acrylic column, and the flow-though was collected at 3 ml/min. The WGA resin was washed with two 50-ml batches of 5 mM MgCl in D/C-EI buffer, one 50-ml batch of D/C-EB (1 mM EDTA, 25 mM bicine, pH 9) buffer, and eluted with two 50-ml and one 20-ml batch of 4 mM chitotriose in D/C-EB buffer.

The WGA eluate (120 ml) was adsorbed onto hydroxylapatite (5.5 g of Bio-Gel HTP (Bio-Rad) equilibrated with D/C-EB buffer) in a 3.1 cm 16-cm acrylic column. The WGA eluate was applied at 1 ml/min, washed with 5 ml of D/C-EB buffer, and eluted at 1 ml/min by sequential addition of 30 ml of 0.12 M potassium phosphate, 1 mM EDTA, D/C, pH 9; 30 ml of 0.05 M potassium phosphate, 1 mM EDTA, D/C, pH 7.4; and 60 ml of 0.5 M potassium phosphate, 1 mM EDTA, D/C, pH 7.4. The eluate contained 15-20% pure rPm2R and was collected in a siliconized 120-ml polypropylene bottle.

25 ml of ABT agarose was recycled with 125-175 ml of 1 M NaOH, 125-175 ml of 1 M NaCl, and 750-1000 ml of HO. The resin was gently mixed with the HTP eluate for 18-36 h at 4 °C; transferred to a siliconized 250-ml sintered glass funnel and washed under vacuum with three 25-ml batches of 0.25 M NaCl in D/C-EI buffer; and transferred to a siliconized 50-ml conical centrifuge tube and eluted three times by rotation for 12-18 h with 25 ml of D/C-EI buffer containing 0.5-1 mM hyoscyamine, 0.25 M NaCl, pepstatin A, and phenylmethylsulfonyl fluoride followed by centrifugation for 20 s for eluate recovery. Combined ABT eluates were dialyzed against two changes of 1 liter of EI buffer plus 25 ml of 10 D/C to reduce the NaCl concentration to below 25 mM. The dialysate was transferred to a siliconized 120-ml polypropylene bottle and adsorbed to 1 ml of TSK-Gel Toyopearl DEAE-650M per 4000 pmol of HTP fraction receptor sites by mixing for 2 h at 4 °C. The resin was transferred to a 3.1 16-cm acrylic column, washed with 200-300 ml of digitonin or digitonin/cholate buffer containing <20 mM salts to remove the remaining hyoscyamine, and the purified rPm2R was eluted with detergent buffer containing 0.25 M NaCl or potassium phosphate, pH 7.4. Sephadex G25F spin columns were used for desalting, with 80-90% recoveries. For long term storage the purified receptor was frozen at -80 °C in siliconized microfuge tubes. About 5% of the receptor activity was lost with one freeze-thaw cycle.

Ligand Binding

[H]QNB binding activity was analyzed using the DEAE filter disc assay (25) with nonspecific binding determined in the presence of 10-100 µM hyoscyamine. The dissociation constant for [H]QNB binding to the receptor, K, was determined by Scatchard analysis(26) . Binding parameters for nonradioactive ligands were determined from competition experiments against [H]QNB. The competition binding data were fit to a model consisting of either one (antagonist) or two (agonist) subclasses of noninteracting binding sites as described(25, 27) .

RQ/R is the fractional saturation of receptor with [H]QNB, and [Q] and [I] are the concentrations of free [H]QNB and inhibitor, respectively. [I] was assumed to be equal to [I] since no displacement was observed until [I] > [R], and the data were normalized to the fractional saturation in the absence of inhibitor. K and K are the dissociation constants for the higher and lower affinity agonist sites, respectively, with F and F = 1 - F their corresponding fractional amounts.

The receptor was diluted into either D/C-EP (1 mM EDTA, 10 mM potassium phosphate, pH 7.4) buffer or D/C, MgBB (5 mM MgCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.4) for ligand binding studies. Variations on these conditions are noted in the table and figure legends. In competition experiments final buffer concentrations were reduced by 25%, and calculated [Mg](28) was reduced to about 3 mM.

Reconstitution

Purified rPm2R or atrial Pm2R was reconstituted with purified atrial G as described(27) . Coupling was assessed by measuring the muscarinic receptor stimulated GTPase activity of G, determined by the difference in GTPase activity between 20 mM carbachol and 10 µM hyoscyamine at 32 °C (27) . Purified rPm2R was also reconstituted into lipids by the same procedure but without added G to assess ligand binding parameters between detergent and lipid environments.

Circular Dichroism

Purified rPm2R was prepared for CD analysis by elution of the DEAE resin with 0.25 M sodium phosphate, pH 7.4, in CD buffer (0.1% digitonin, 0.02% cholate, 0.2 mM EDTA, 10 mM potassium phosphate, pH 7.4), and the excess phosphate was removed by gel filtration. The receptor concentration was 1.4 µM in [H]QNB sites with a specific activity of 14.4 nmol/mg of protein. Protein content was measured by analysis for total amino acids (23) in order to directly determine the total concentration of amide bonds. Buffer controls were prepared in parallel with the receptor preparation. rPm2R was incubated for 1 h at room temperature with 1 mM acetylcholine, 1 mM carbachol, 20 µM oxotremorine M, 100 µM (+)-pilocarpine, 10 µM(-)-hyoscyamine, or 10 µM QNB and stored for 1-2 days at 4 °C before CD analysis. The CD of each sample was measured in 100-micron path length cells from 260 to 178 nm with total absorbance less than 1.0 to ensure sufficient light transmission. Spectra were measured at 20 °C using a model J-720 Jasco spectrometer calibrated with (+)-10-camphorsulfonic acid at two points to ensure reliability(29) . The data were collected at 1-nm intervals using an on-line PC computer, and at least 6 spectra for each sample were averaged, corrected by subtraction of buffer spectra (usually negligible), and then smoothed. Secondary structure was analyzed for -helix, antiparallel -sheet, parallel -sheet, -turn, and random coil by the variable selection method(30) .


RESULTS

Purification

Table 1shows a representative purification. Final receptor yield was 16% of the total homogenate activity and over 30% of that for isolated membranes. The final specific activity was 11.9 (± 2.0) nmol/mg of protein (average of four preparations), the same specific activity obtained for the purified atrial Pm2R(5) . The yield of purified CHO cell rPm2R protein was 100-fold greater than for the purified atrial Pm2R. A specific activity of 12.5 nmol/mg corresponds to a mass of 80 kDa for the receptor, assuming one ligand binding site per receptor molecule. The rPm2R preparation consisted of a single diffuse silver-stained band on SDS-polyacrylamide gel electrophoresis, similar to that of the atrial Pm2R (Fig. 1, and (5) ), with a mass of 78.5 kDa (range 60-100 kDa) for the rPm2R and 80 kDa (range 60-112 kDa) for the atrial Pm2R when analyzed together(32) . Thus, the rPm2R and atrial Pm2R preparations were purified to homogeneity with full retention of ligand binding capacity. Atrial and recombinant preparations were stable on ice for a month or more, and considerably longer at -80 °C, with only small losses (5%) in binding activity associated with a freeze-thaw cycle.




Figure 1: Silver-stained SDS-polyacrylamide gel of the fractions obtained during purification of rPm2R from CHO cells. The gel was an 8-18% polyacrylamide gradient in a minigel format using the discontinuous buffer system of Laemmli(33) . Lane1, molecular mass standards (phosphorylase a, M 97114; bovine serum albumin, M 66296; ovalbumin, M 42807; aldolase, M 39210; concanavalin A, M 25271; soybean trypsin inhibitor, M 20095; lysozyme, M 14314); lane2, 1 µg of homogenate; lane3, 1 µg of membranes; lane4, 1 µg of extract; lane5, 1 µg of WGA eluate; lane6, 0.5 µg of HTP eluate; lane7, 0.1 µg of ABT eluate. The gel was silver-stained according to the method of Wray et al.(34) .



The CHO cell receptor preparation was very susceptible to proteolysis, which could not be eliminated even by judicious use of a wide spectrum of protease inhibitors. Speed of processing during the early stages of the preparation was essential to successful isolation of intact receptor. Despite the high specific activity of the membranes that could be isolated from the overexpressing CHO cells, we were unable to achieve purification of the rPm2R without the WGA and hydroxylapatite chromatography steps. Membranes from CHO cells banded at a higher sucrose density than those from atrial tissue, and unlike the atrial Pm2R (25) no purification was achievable during solubilization. The CHO cell rPm2R bound to WGA agarose more tightly than did the atrial receptor and required the use of chitotriose for elution. The hydroxylapatite and ABT affinity purification steps gave results similar to those found for the atrial preparation.

Equilibrium Ligand Binding

Representative Scatchard plots for the binding of [H]QNB to the purified rPm2R and atrial Pm2R are shown in Fig. 2. For three different preparations incubated for 24 h at room temperature (18-22 °C) in D/C-EP buffer the apparent K for [H]QNB binding was 98 ± 26 pM for the recombinant receptor and 76 ± 8 pM for the atrial receptor. Agonist interactions with the purified receptor were best fit to a model predicting two subclasses of sites, and conditions affecting agonist binding were examined in the carbachol competition experiments described below. Under similar conditions the carbachol binding parameters were essentially the same for both the purified rPm2R and atrial Pm2R.


Figure 2: Binding of [H]QNB to purified Pm2R. Purified recombinant Pm2R and atrial Pm2R were diluted into D/C-EP buffer and incubated with various concentrations of [H]QNB for 24 h at room temperature (18-22 °C). Nonspecific binding was determined by preincubation with 10 µM hyoscyamine for 20 min prior to addition of [H]QNB. The binding data were analyzed by the method of Scatchard (26) (inset) and fitted by linear regression. The lines represent the fitted curves, which gave K values of 82 ± 10 pM and 83 ± 5 pM for the recombinant and atrial receptors, respectively.



Reconstitution of Purified Pm2R and Purified G

The ability of purified rPm2R to couple to G in a reconstituted system was compared with that for the purified atrial Pm2R(27) . The results of these studies are shown in Table 2. Similar levels of incorporation were obtained for both receptor preparations (30%), and there were no significant differences in their ability to couple to the purified atrial G proteins. Under parallel conditions, both receptor preparations showed similar levels of agonist-stimulated GTPase activity.



Conditions Affecting Ligand Binding

Fig. 3shows the effects of Mg on carbachol binding. At 20 °C and 0.075% digitonin, increasing Mg had the effect of shifting the carbachol competition curves leftward (panelA), due in large part to an increase in F from zero in the absence of Mg to 30% in the presence of 18.75 mM MgCl (Table 3). At 4 °C and higher detergent concentration, increasing Mg also caused a leftward shift of the titration curve, but in this case changes in F were small (panelB). Instead, K was reduced about 6-fold when Mg was present. K was reduced about 3-fold in the presence of Mg under both experimental temperature/detergent conditions. There was no apparent affect of Mg on K. Effects of Mg on the purified atrial Pm2R were similar (data not shown).


Figure 3: Effect of [Mg] on the carbachol titrations of purified rPm2R. Total binding site concentrations were about 400 pM, and total [H]QNB was about 750 pM. PanelA experiments were done with receptor diluted into BB containing 0.075% digitonin (final) and the indicated final concentrations of MgCl. Incubations were for 18-20 h at room temperature (18-22 °C). PanelB experiments were done in the presence of BB and 0.75 D/C (final) and the indicated final concentrations of MgCl. Incubations were for 70 h at 4 °C. The fitted binding parameters are given in Table 3.





Detergent effects on carbachol binding to the purified rPm2R are shown in Fig. 4A. The competition curves were slightly left-shifted when the detergent concentration was lowered to 0.075% digitonin alone. Under conditions of reduced detergent, K and K were reduced, and F was increased (Table 4). These differences were greater at 20 °C than that at 4 °C. With the purified atrial Pm2R, lower detergent concentrations also reduced Kand K, but effects on F were more dramatic. The K site of the atrial receptor was undetectable in experiments carried out with 0.3% digitonin, 0.15 D/C, or 0.75 D/C in MgBB for 21 h at 25 °C but was clearly observable with 0.075% digitonin (F = 26 ± 6%).


Figure 4: Effect of detergent concentration and temperature on the carbachol titrations of purified rPm2R. Total binding site concentrations were about 400 pM, and total [H]QNB was about 750 pM. PanelA depicts the effects of detergent under two different conditions: 18.75 mM MgCl at 20 °C (circles) and 3.75 mM MgCl at 4 °C (squares). PanelB depicts the effect of temperature with the receptor in 18.75 mM MgCl in 0.75 D/C, BB. The fitted binding parameters for these experiments are given in Table 4.





Fig. 4B shows the effect of temperature on the carbachol displacement curves for the purified rPm2R in 18.75 mM MgCl, D/C, BB (10 mM HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.4). The carbachol curves were left-shifted 1 order of magnitude from 37 to 20 °C and again from 20 to 4 °C. As shown by the fitted binding parameters for these experiments (Table 4), this leftward shift resulted from a decrease in K and K and an increase in F. These same trends were observed under the conditions of low detergent and 3.75 mM Mg for both the recombinant and atrial Pm2R (data not shown). The carbachol competition curves done at 4 °C in either 18.75 mM MgCl, 0.75 D/C, BB or 3.75 mM MgCl, 0.075% digitonin, BB were superimposible.

The effects of temperature on carbachol/[H]QNB competition experiments on purified rPm2R following detergent removal by reconstitution into phospholipid vesicles were similar to that for the receptor in detergent. The displacement curves were left-shifted with decreasing temperatures and appeared much like those of Fig. 4B, except that the corresponding shifts were slightly smaller. The fitted binding parameters for the reconstituted preparations are given in Table 4and are similar to the receptor in detergent except that K was less temperature-sensitive. The overall effects of decreasing temperature, namely, decreasing K and K, increasing F, and no detectable K at 37 °C, was observed for purified receptor in both detergent and liposomes.

Pm2R Secondary Structure from Circular Dichroism

Representative CD spectra for the purified rPm2R are shown in Fig. 5. These spectra showed strong negative deflections with minima at 220 and 208 nm and a strong maximum positive deflection at 190 nm indicative of the -helical structure. Analysis of the spectrum for the nonliganded receptor indicated 53% -helix (Table 5). Addition of agonists to the receptor (Fig. 5, A and B) produced little change in the CD spectra, whereas antagonists (Fig. 5, C and D) showed more negative deflections at the 220 and 208 nm minima. In all CD experiments, the QNB-receptor complex always produced deeper negative deflections at 220 and 208 nm than either the nonliganded or agonist liganded receptor. Secondary structural analysis of the QNB-liganded receptor spectrum showed a marginally significant 4% decrease in -helix and 4% increase in parallel -sheet structure compared with the free receptor (Table 5).


Figure 5: CD spectra of the purified rPm2R. Aliquots of one preparation of purified rPm2R preparation (14.4 nmol of [H]QNB sites/mg of protein) were incubated with ligands, and the CD spectra were collected as described under ``Experimental Procedures.'' The spectra for the agonist oxotremorine M and the partial agonist pilocarpine are not shown but were identical to the acetylcholine and carbachol spectra. In each panel the solidline represents the spectrum of the free rPm2R and the dashedline the spectrum for the receptor plus ligand. Spectral data were analyzed on a per amide basis calculated from protein content and sequence ( is in units of M cm). Analyses of secondary structures are given in Table 5for the free receptor and QNB-receptor complex (panelD).






DISCUSSION

Purification of the rPm2R from a clonal line of CHO cells overexpressing this receptor resulted in a preparation of 2 mg of homogeneous receptor protein at theoretical specific activity. The yield is nearly 100-fold greater than the yield of purified atrial Pm2 receptor(5) . The purification of rPm2R also compares favorably with the rHm2R preparations from the baculovirus-infected insect Sf9 cell system(14) . These preparations of recombinant m2R will permit more extensive characterization of the purified receptor and provide a model system to advance our understanding of the molecular structure and function of G protein-coupled receptors in general.

In most respects the purified rPm2R was indistinguishable from the purified atrial Pm2R. Ligands bound to both preparations with the same affinities. Both preparations coupled to purified G proteins with the same efficiency in reconstitution experiments. Carbachol/[H]QNB competition assays were similarly affected by Mg, detergent, and temperature in both preparations. There were only slight differences in SDS-polyacrylamide gel electrophoresis migration between the recombinant and atrial receptors, suggesting that their overall extent of glycosylation was similar. The recombinant receptor did bind more tightly to WGA than the atrial receptor, indicating that it differs in the amount of surface-exposed sialic acid residues. The mass of the rHm2R prepared from the insect cell system was 55 kDa on SDS-polyacrylamide gels(14) , which is considerably smaller than the 80-kDa mass found for the Pm2R preparations and indicates that glycosylation of recombinant m2R in the insect cell system is different from the atrial or CHO cell system.

Agonists compete with [H]QNB for more than one subclass of sites in the purified mAcChR (5, 16, 17) and adenosine receptor (13) , but the significance of two agonist affinity states in the purified receptor has not been addressed. Agonist competition curves for the membrane-bound rPm2R can be analyzed in terms of three agonist affinity states(36) , similar to natural tissue sources(37, 38, 39) . The highest affinity site (K about 3 nM for carbachol) is sensitive to guanine nucleotides, indicating it is the site associated with activation of G proteins. The two remaining membrane agonist affinity states bind carbachol with an apparent K of 1.6 and 30 µM.()These K values are within range of the two carbachol affinity states of the purified rPm2R and atrial Pm2R in detergent as well as in phospholipids after reconstitution and may represent the same receptor states. Moreover, the common characteristics found for the two G protein-independent agonist affinity states, K and K, among membrane-bound, detergent-solubilized, and phospholipid-reconstituted pure receptor demonstrate that the purified mAcChR in detergent is a good system for further probing these states.

Mg in the 1-10 mM range is needed for hormone activation of G proteins and differs from the high affinity (nM) Mg site associated with G protein GTPase activity(40) . The low affinity Mg site(s) is required for formation of nucleotide-sensitive high affinity agonist binding and for dissociation of activated G protein into its and subunits(41, 42, 43) . Formation of this site appears to be primarily at the expense of depletion of the intermediate affinity agonist site(36, 38, 42) . Mg effects on the purified Pm2R ( Fig. 3and Table 3) demonstrate that mM Mg also promotes formation of the intermediate affinity agonist state. Thus mM Mg may play a regulatory role in stabilizing the intermediate affinity agonist conformation prior to G protein interaction.

The leftward shift of the carbachol competition curves at lower temperatures was due to a large increase in the proportion of the receptor in the high affinity agonist state (F) and to a decrease in K. Low temperature-induced increases in agonist affinity were reported in studies of the membrane-bound mAcChR(44, 45, 46) , and adrenergic receptors(47, 48, 49, 50) , dopamine receptors(51) , and in the detergent solubilized preparations of these receptors (47, 50) as well as in intact cells (50, 52) and membranes in which G proteins were removed by alkalinization or proteolysis(53, 54) . In two studies(49, 50) , the major low temperature effects were attributed to an increase in F while K remained unchanged. The above results show that this property also persists in purified receptor preparations either in detergent or in liposomal environments, demonstrating that it is an intrinsic characteristic of the receptor.

K and K may be characterized by similar lipid-receptor interactions, which are not shared with K. Low concentrations of detergents and low temperatures favored decreases in both K and K, while K was largely unaffected. Detergent effects were more prominent at 20 than at 4 °C, and differences were observed between the recombinant and atrial preparations, perhaps because of different boundary lipid compositions. K and K for the purified rPm2R in both detergent and phospholipid were greater than their corresponding membrane-bound values, but K was essentially identical under all such conditions. The large temperature effects on K and K indicate that ligand binding energies for these interactions involve large enthalpy changes, but K was unaffected by temperature (H 0), and the favorable standard free binding energy G = -8.2 kcal/mol (for K 1 µM) must arise from mostly entropic contributions (S = +27.4 cal/deg mol), a feature characteristic of hydrophobic interactions(55) .

The CD spectra demonstrated that little change in secondary structure was associated with agonist occupancy of the purified rPm2R at room temperatures, but small changes were observed with antagonist binding. Ligand-receptor interactions may not require large changes in secondary structure, but the results observed with QNB were reproducible. At this time, it is not clear how the two agonist affinity states relate to the CD measurements, but if there are two distinct agonist-receptor conformations, the CD spectrum should be a weighted average of the two. The overall secondary structure analysis of the CD spectra are consistent with the putative seven-transmembrane-spanning structure for the mAcChR. These domains account for about 36% of the -helical content. Thus the remaining 17% suggests that the intra- and extracellular domains must also contain a significant amount of -helix. The secondary structure analysis for the rPm2R was found to be very similar to that reported for the related G protein-coupled membrane protein rhodopsin analyzed by the same method (35) and by Fourier transform infrared spectroscopy(56) , which gave an estimated 51% -helical content.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL23632 and ES00210 (to M. I. S.) and GM21479 (to W. C. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 503-737-2029; Fax: 503-737-0481.

The abbreviations used are: mAcChR, muscarinic acetylcholine receptor; G proteins, guanine nucleotide binding proteins; m2R, muscarinic acetylcholine receptor subtype 2; Hm2R or Pm2R, human and porcine m2R, respectively; rPm2R, recombinant Pm2R; CHO, Chinese hamster ovary; dhfr CHO cells, CHO cells deficient in dihydrofolate reductase; QNB, R-(-)-quinuclidinyl benzilate; WGA, wheat germ agglutinin; ABT, 3-(2`-aminobenzhydryloxy)tropane; HTP, hydroxylapatite; CD, circular dichroism; D/C, 0.4% digitonin, 0.08% cholate; BB, binding buffer; CHM, cell-homogenizing medium; GTPS, guanosine 5`-O-(thiotriphosphate).

G. L. Peterson and M. I. Schimerlik, unpublished observations.


ACKNOWLEDGEMENTS

We acknowledge Dr. Daniel Capon of Genentech, Inc. for providing the pSVE expression system and the clone of the Pm2R gene, and we thank Bipei Chen for technical assistance.


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