Double Mutant Cycle Analysis of Aspartate 69, 97, and 103 to Asparagine Mutants in the m2 Muscarinic Acetylcholine Receptor

Archives of Biochemistry and Biophysics Vol. 361, No. 2, January 15, pp. 283–294, 1999 Article ID abbi.1998.0985, available online at http://www.idealibrary.com on

Double Mutant Cycle Analysis of Aspartate 69, 97, and 103 to Asparagine Mutants in the m2 Muscarinic Acetylcholine Receptor Walter K. Vogel, Gary L. Peterson, David J. Broderick, Valerie A. Mosser,* and Michael I. Schimerlik* ,1 Department of Biochemistry and Biophysics and *Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331-7305

Received September 8, 1998

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor. © 1999 Academic Press

1 To whom correspondence should be addressed. Fax: (541) 7370481. E-mail: [email protected].

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

Key Words: G protein-coupled receptors; m2 muscarinic acetylcholine receptors; site-directed mutagenesis; mutant analysis.

Muscarinic acetylcholine receptors are members of a diverse superfamily of receptors that are characterized structurally by seven transmembrane helices and functionally by transducing extracellular signals through G proteins. Five muscarinic receptors have been identified and are designated subtypes m1–m5 (1– 4). The m2 subtype is widely distributed in mammalian tissue and is the only subtype found in mammalian heart (3), where it inhibits adenylyl cyclase and stimulates both inwardly rectifying potassium channels and phospholipase C via pertussis toxin-sensitive G proteins. Cardiac muscarinic receptor activity slows the rate and decreases the force of contraction of the heart by at least two mechanisms. Voltage-gated calcium currents, regulated via cAMP levels, are reduced and inwardly rectifying potassium currents are increased (5). In the absence of high-resolution structures of G protein-coupled receptors, most insights into their structure and function have been inferred from chemical modification and site-directed mutagenesis experiments and rhodopsin projection structures. The projection structures (6, 7) resolved the transmembrane domain into seven a-helices and placed helices 2 and 3 close together. Site-directed mutagenesis experiments suggested that conserved motifs found at the intracellular ends of these helices (8) have roles in G protein activation (9, 10) and residues located near the extracellular end of helix 3 affect agonist binding affinity (11–15). Muscarinic receptors have conserved aspartate residues in these motifs, one at the cytoplasmic 283

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end of helix 2 (Asp69 in the m2 muscarinic receptor) and two near the extracellular end of helix 3 (Asp97 and Asp103). The effects of aspartate to asparagine mutations at these sites on the free energy of ligand binding, signal transduction, and receptor thermal stability were examined in single and double mutant combinations. The double mutant cycle approach is often used to determine if the effects of two mutations are independent or whether a mutation at one site alters the properties of an amino acid at a second site. For example, if two mutations have independent effects on ligand binding properties, standard free energy changes for ligand binding (DDG9°) should be additive and the sum of DDG9° values for the two single mutants versus the wild type should equal that of the double mutant compared to the wild type. If this is not true, it would argue that a perturbation caused by the mutation has been transmitted through the protein structure such that the properties of the amino acid at the second site have been affected. Helices 2 and 3 are thought to be in close contact, and, presumably, during signal transduction information is transferred from the ligand binding site near Asp97 and Asp103 to regions involved in G protein coupling (Asp69). Thus, by utilizing both single and double mutants it was possible not only to determine the results of each single mutation but to ask whether each individual mutation has affected the properties of amino acids at other locations within the m2 muscarinic receptor structure. To the best of our knowledge, this is the first application of double mutant cycle analysis to a G protein-coupled receptor. MATERIALS AND METHODS Materials. 3H-labeled R-(2)-quinuclidinyl benzilate (QNB) 2 [ H]NMS, [ 3H]oxotremorine M, and [ 14C]cAMP were purchased from DuPont-NEN Research Products (Boston, MA). [ 3H]Adenine and myo-[ 3H]inositol were from American Radiolabeled Chemicals (St. Louis, MO). Acetylcholine and gallamine were from Sigma (St. Louis, MO). Carbachol and pilocarpine were from Aldrich (Milwaukee, WI). Oxotremorine M and pirenzepine were from Research Biochemicals (Natick, MA). Gpp(NH)p was from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Ro 20-1724 was from BIOMOL Research Laboratories (Plymouth Meeting, PA). Tissue culture medium was from Gibco/BRL (Gaithersburg, MD). The pcDNA3 expression vector was from Invitrogen (Carlsbad, CA). The pSVE expression vector was kindly provided by Genentech (South San Francisco, CA) and AF-DX 116 was a kind gift from Boehringer-Mannheim Biochemicals. 3

2 Abbreviations used: CHO, Chinese hamster ovary; DxN, an aspartate to asparagine mutation where the number x indicates the position that mutation was introduced; Dx.yN, an aspartate to asparagine double mutant where x and y indicated the two positions that mutations were introduced; Gpp(NH)p, guanylylimidodiphosphate; NMS, S-(2)-N-methylscopolamine; QNB, R-(2)-quinuclidinyl benzilate; DMEM, Dulbecco’s modified Eagle’s medium; F12, Ham’s F-12; K L, K M, and K N, the low-, medium-, and high-affinity dissociation constants, respectively.

Tissue culture. CHO cells deficient in dihydrofolate reductase (16) were transfected by the calcium phosphate method (17) with the pSVE expression vector (2) containing the coding region of the porcine m2 muscarinic acetylcholine receptor. The pSVE expression vector contains a mouse dihydrofolate reductase gene that permits the establishment of stable transfected cell lines under the selective pressure of methotrexate (0.5–5 mM). Clonal cell lines were grown in Dulbecco’s modified Eagle’s media/Ham’s F-12 (DMEM/F12) minus glycine, hypoxanthine, and thymidine, plus 10% dialyzed calf serum and 10 mg/ml insulin. Cell lines were adapted to spinner culture in this medium except with 1 mg/ml insulin. For large-scale preparations, CHO cells were grown in DMEM/F12 plus 10% calf serum without selective pressure for 3– 4 days before harvesting to allow more rapid growth. The addition of sodium butyrate (5 mM), in the last 12–24 h before harvesting, increased receptor expression two- to nine-fold (18). CHO K1 cells, grown in DMEM/F12 plus 10% calf serum, were transfected by the lipofectase method with pcDNA3 expression vector containing D69.103N mutant muscarinic receptor gene. These transfected clonal CHO K1 cells were maintained under the selective pressure of 400 mg/ml geneticin. Site-directed mutagenesis. The aspartate mutant m2 muscarinic receptors D69N, D97N, D103N, and D97.103N were produced by oligonucleotide-directed mutagenesis according to the method of Kunkel et al. (19), whereas D69.97N and D69.103N were produced according to the method of Vandeyar et al. (20). The double mutants were produced by sequential mutagenesis from single mutant templates. The wild-type coding region was ligated into M13mp18 as a HindIII/EcoRI fragment derived from the pSVE expression vector (2). The mutations were introduced by priming second-strand synthesis with the antisense oligonucleotides: 59-GATGAGGTTAGCACAGGC-39 (D69N), 59-CCAAAGGTTACACACCAC-39 (D97N), and 59-CACGTAGTTCAGAGCTAG-39 (D103N) (base changes are underlined). Mutant sequences were confirmed by dideoxy sequencing (21). Ligand binding. Ligand binding to sucrose gradient-enriched membrane preparations (22, 23) was assessed in 10 mM Hepes, 5 mM MgCl 2, 1 mM EDTA, 1 mM EGTA, pH 7.4, at 25°C. After 4 – 6 h samples were filtered through Schleicher & Schuell (Keene, NH) No. 32 glass-fiber filter membranes, pretreated with 0.1% (w/v) polyethylenimine, on a Brandel harvester (Gaithersburg, MD) and washed with 10 ml ice-cold 50 mM sodium phosphate, 1 mM EDTA, pH 7.4. Bound radiolabel was determined by liquid scintillation counting. Filter binding assays on D103N-containing mutants were done on samples that were chilled on ice briefly prior to a 6-ml wash with ice-cold buffer. Nonspecific binding was assessed in the presence of excess unlabeled ligand. NMS and QNB binding results were from the direct binding of the radiolabeled ligand except for the D97.103N mutant, for which NMS affinity was determined by competition with [ 3H]QNB. Other ligand affinities were determined by competition with either [ 3H]NMS or [ 3H]QNB or both. Agonist competition binding experiments were conducted in the presence and absence of Gpp(NH)p, a nonhydrolyzable analog of GTP, and the data were fit so that values for K H, K M, and K L were shared in the analysis. Protein stability. Resistance to thermal inactivation was assessed in enriched membrane preparations in the presence and absence of saturating [ 3H]QNB ($10-fold K d). Membranes were exposed to different temperatures for 1 h and quenched on ice, and the residual [ 3H]QNB binding was determined. Effector coupling. Phospholipase C stimulation was determined in attached CHO cells expressing wild-type and mutant muscarinic receptors plated at 2 3 10 4 cells/mm 2 and incubated in 4 mCi/ml myo-[ 3H]inositol for 72 h. Agonists were added and inositol 1-phosphate accumulation was measured after 30 min at 37°C as described by Lee et al. (24). Inhibition of forskolin-stimulated adenylyl cyclase activity was determined in attached CHO cells in the presence of 100 mM isobutylmethylxanthine, 50 mM Ro 20-1724, and agonist after exposing the cells to 8 mCi/ml [ 3H]adenine for 2–3 h. The 20-min 37°C

DOUBLE ASPARTATE MUTAGENESIS IN THE m2 MUSCARINIC RECEPTOR assay was started by the addition of 40 mM forskolin, and the assay product [ 3H]cAMP was determined according to the method of Salomon (25). To compare assay results at similar receptor densities, wild-type receptor levels were reduced in some experiments by incubating the cells 1 h at 37°C with varying concentrations of the slowly dissociating antagonist QNB (26). Data analysis. Functional assays were fit to Eq. [1], where max 1 and max 2 are the maxima of the inhibitory and stimulatory portions of the curve, min is the minimum of the curve, [x] is the agonist concentration, and p1 and p2 are the respective slope factors. For adenylyl cyclase assays that showed only an inhibitory phase the first two terms were used, and for assays of phospholipase C stimulation the first and last terms were used. CPM 5 min 1

max1 2 min @x# 11 ~EC50 i!

S

D

p1

1

max2 2 min ~EC50 s! 11 @x#

S

D

p2

[1]

In direct saturation binding experiments, total bound radioligand was analyzed as the sum of specifically, [RL], and nonspecifically, N[L], bound ligand according to the quadratic solution of Eqs. [2] and [3], where [R 0 ] and [L 0 ] are the total receptor and total radioligand concentrations, respectively; K is the equilibrium dissociation constant; and N is the proportionality constant of nonspecifically bound radioligand to free radioligand concentration. @RL# 5

~@R0 # 2 @RL#!~@L0 # 2 @RL#! K~N 1 1!

[2]

N@L# 5

N~@L0 # 2 @RL#! N11

[3]

Unlabeled agonist and antagonist binding was assessed by competition with radiolabeled antagonist. Agonist binding data were fit to one to three noninteracting classes of sites that bound radiolabeled antagonist with the same dissociation constant (Eq. [4]). Total specifically bound radiolabeled antagonist, [RL], is the sum of that bound at each subclass of agonist binding sites according to the polynomial solutions to Eq. [4]

O n

@RL# 5

i51

F i @R0 #~@L0 # 2 @RL#! , @A# K~N 1 1! 1 1 1 @L0 # 2 @RL# Ki

S

D

[4]

where [R 0] is the total concentration of receptor, F i is fraction of [R 0] at site i, [L 0] is the total radiolabeled antagonist concentration, [A] is the total agonist concentration (equal to the free concentration since no displacement occurs until [A] . [R 0]), K is the dissociation constant for radiolabeled antagonist, and K i is the dissociation constant for the agonist at site i (referred to as K H, K M, and K L, the high-, medium-, and low-affinity dissociation constants, respectively). Data were fit by nonlinear least-squares procedures using Marquardt’s algorithm (27), and parameter values are reported as the mean and 95% confidence interval of several determinations (P 5 0.05). Ligand binding energetics. The difference in standard free energies for ligand binding between mutant and wild-type muscarinic receptors was calculated according to Eq. [5] as DDG9° 5 DG9°Mutant 2 DG9°Wild-type 5 RT ln~K Mutant/K Wild-type),

[5]

where DG9° is the standard Gibbs free-energy change, R is the gas constant, T is the absolute temperature, and K is the equilibrium dissociation constant. DDG9° is the apparent difference in the bind-

285

ing energy contribution between the carboxylate anion of aspartate and the neutral asparagine amide (28); it does not take into account the changes in solvation between the wild-type and the mutant receptor or potential changes in receptor stability. DDG9° is defined such that positive values indicate that the mutant binds ligand more weakly than the wild type.

RESULTS

Mutagenesis and Expression Three aspartate residues located in the transmembrane a-helices 2 and 3 of the m2 muscarinic receptor were mutated individually and in pairs to asparagine. Experiments on D103N- and D69.103N-expressing clonal CHO cell lines were hampered by poor expression of antagonist binding sites. (D103E and D103C were also poorly expressed; D. J. Broderick and M. I. Schimerlik, unpublished data.) Similar problems precluded the characterization of the D103N homologous mutation in the m1 muscarinic receptor (9). Limited ligand binding data on the D69.103N mutant were obtained from CHO cells transfected with an alternative expression vector (pcDNA3). The results from this approach were poor and not further pursued to obtain D103N-expressing cell lines. The effect of the D103N mutation was characterized by comparing the double mutants D69.103N and D97.103N with the single mutants D69N and D97N. Binding Studies Ligand binding characterization of the D103N-containing mutants was complicated by the low-affinity and high-dissociation rate of the radiolabeled antagonists. At 0°C the rate constants for QNB and NMS dissociation from the D97.103N mutant were approximately 5 3 10 23 s 21 and 2.5 3 10 22 s 21, respectively (data not shown). D103N-containing mutants were thus characterized with QNB via a slightly altered filter binding procedure (see Materials and Methods). QNB affinity of D103N-containing mutants was also determined using an air-driven ultracentrifuge to separate bound from free ligand. These results were indistinguishable from those obtained by the modified filter binding assay. The results of the ligand binding studies are presented in Table I. Antagonists AF-DX 116, hyoscyamine, NMS, pirenzepine, and QNB bound to a single class of sites in both the wild-type and mutant receptors. Gallamine bound to both a competitive site and an allosteric site that has the effect of reducing the dissociation rate of other ligands (29); only the affinity at the competitive site was determined. Agonists bound to three classes of sites in membrane preparations containing the wild-type m2 muscarinic receptor. The highest affinity site, K H, was sensitive to guanine nucleotide (Fig. 1), consistent with agonist binding to a

286

VOGEL ET AL. TABLE I

Equilibrium Dissociation Constants for Wild-Type and Mutant m2 Muscarinic Receptors Units Antagonists NMS pM QNB pM AF-DX 116 nM Pirenzepine nM Hyoscyamine nM Gallamine nM Agonists—K H Acetylcholine nM Carbachol nM Oxotremorine M nM Pilocarpine nM Agonists—K M Acetylcholine mM Carbachol mM Oxotremorine M mM Pilocarpine mM Agonists—K L Acetylcholine mM Carbachol mM Oxotremorine M mM Pilocarpine mM

Wild-type

D69N

253 6 12 (15) 17.4 6 0.7 (16) 211 6 39 (7) 280 6 66 (8) 1.42 6 0.23 (12) 49.1 6 4.7 (7)

297 6 17 (13) 18.6 6 0.9 (13) 209 6 63 (4) 700 6 140 (6) 0.95 6 0.10 (4) 379 6 86 (5)

1.45 6 0.15 (12) 3.6 6 0.19 (19) 2.16 6 0.29 (10) 27.2 6 6.2 (8)

D97N

D69.97N

272 6 20 (6) 675 6 54 (5) 96.3 6 3.7 (14) 116 6 20 (7) 676 6 130 (2) 227 6 133 (3) 720 6 58 (4) 1280 6 149 (3) 2.11 6 0.29 (3) 5.7 6 2.7 (3) 49.2 6 4.0 (4) 110 6 42 (2)

D69.103N

32,500 6 3700 (3)

D97.103N 32,600 6 2550 (9) 12,200 6 600 (13) 17,000 6 3000 (5) 9000 6 800 (5) 110 6 18 (5) 1700 6 300 (5)

34 6 15 (6) 14.7 6 4.8 (4) 13.4 6 3.9 (4) 266 6 60 (3)

0.35 6 0.02 (15) 1.37 6 0.03 (38) 0.40 6 0.04 (16) 2.78 6 0.20 (14)

0.66 6 0.07 (8) 3.45 6 0.44 (9) 3.47 6 0.18 (5) 2.24 6 0.34 (9)

11.1 6 1.1 (13) 21.7 6 0.6 (37) 13.0 6 2.9 (9) 24.9 6 5.5 (9)

13.0 6 3.8 (6) 25.7 6 4.6 (5) 20.2 6 3.6 (5) 43 6 14 (4)

7.8 6 2.4 (6) 1.85 6 0.75 (4) 22,700 6 11,000 (3) 3.4 6 1.3 (7) 8.1 6 1.2 (7) 30,000 6 120,000 (2) 5.0 6 2.2 (2) 3.2 6 1.4 (4) 16,700 6 15,100 (2) 5.3 6 1.4 (3) 5.0 6 2.1 (3) 740 6 4200 (2) 116 6 43 (6) 101 6 20 (7) 370 6 1250 (2) 350 6 170 (3)

34.7 6 5.6 (4) 110 6 10 (8) 269 6 90 (4) 120 6 120 (3)

21,700 6 5,200 (5) 8800 6 500 (16) 1250 6 110 (11) 1038 6 54 (7) 183,000 6 27,000 (8) 133,000 6 143,000 (2) 34,000 6 11,000 (4)

Note. Values are means 6 95% confidence interval (P 5 0.05); the number of experimental determinations is indicated in parentheses.

receptor–G protein complex. The medium affinity site, K M, appeared to be the free receptor since its relative proportion increased in the presence of guanine nucleotides. The concentration of the lowest affinity site, K L, was not affected by guanine nucleotides and its role has not been assigned (26). Mutant constructs incorporating D69N and/or D103N did not display high-affinity guanine nucleotide-sensitive agonist binding in competition binding experiments nor was it detectable in the D69N mutant by direct [ 3H]oxotremorine M binding. Guanine nucleotide-sensitive agonist binding was evident only in the wild type and the D97N mutant (Table I, Fig. 1). Values for DDG9° between the asparagine mutant and the wild-type aspartate derived from the data in Table I are presented in Table II as are the additivity values for D69N, D97N, and D69.97N. The DDG9° values for the D103N mutant were calculated as the difference between the D69.103N and D69N and between D97.103N and D97N. Thermal Inactivation Resistance to irreversible thermal inactivation of ligand binding in the presence and absence of the antagonist QNB was used to estimate the effects of the mutations on the stability of the m2 muscarinic receptor structure. In the absence of QNB, the differences in thermal stability between the wild type and the mutants were small or not significant (Table III). The QNB bound state was stabilized relative to the unli-

ganded state by approximately 6°C in the wild type and the D69N mutant. The D97N mutant and the D69.97N double mutant reduced the QNB stabilization effect to approximately 4°C, whereas the incorporation of D103N (D69.103N and D97.103) eliminated it. These experiments were conducted at saturating QNB concentrations that took into account the reduced QNB affinity of the mutants, suggesting that the effect on stability was not due to reduced affinity. Functional Activity Functional coupling of the mutant receptor was compared to that of the wild-type receptor at similar expression levels. Since wild-type functional coupling varied with receptor density, the receptor density of the wild-type-expressing cell line was reduced with the slowly dissociating antagonist QNB to provide a valid basis of comparison (26). Functional coupling of the wild-type receptor expressed in CHO cells was previously reported (26). The wild-type-expressing cell line stimulated phospholipase C to a maximal extent that varied proportionally with receptor density, whereas the concentration of agonist that produced a half-maximal response (EC 50) was independent of receptor density (Table IV). In contrast, the EC 50 for the inhibition of adenylyl cyclase was inversely proportional to receptor density, whereas the maximal response did not vary significantly with acetylcholine, carbachol, and oxotremorine M. Maximal adenylyl cyclase response

DOUBLE ASPARTATE MUTAGENESIS IN THE m2 MUSCARINIC RECEPTOR

287

FIG. 1. Agonist displacement by oxotremorine M of wild-type and asparagine mutant muscarinic receptors in CHO cell membranes. Data were fit to between one and three independent classes of sites, as described under Materials and Methods. The parameter values for the fitted lines are reported 6 asymptotic standard errors. Wild-type K H 5 3.0 6 0.7 nM, K M 5 0.18 6 0.02 mM, K L 5 7.5 6 1.0 mM, total [ 3H]NMS 5 2.1 nM, total receptor 5 100 pM, F H 5 51.6 6 2.2%, F M 5 30.7 6 2.8%, in the absence of Gpp(NH)p (F), and F H 5 0% (fixed), F M 5 84.2 6 1.6%, in the presence of 100 mM Gpp(NH)p (E). D69N K M 5 8.1 6 0.9 mM, K L 5 166 6 10 mM, total [ 3H]NMS 5 4.1 nM, total receptor 5 500 pM, F M 5 55.7 6 2.2%, in the absence of Gpp(NH)p (F), and F M 5 54.1 6 2.3%, in the presence of 100 mM Gpp(NH)p (E). D97N K H 5 12.9 6 3.1 nM, K M 5 4.8 6 0.3 mM, K L 5 488 6 9 mM, total [ 3H]NMS 5 1.7 nM, total receptor 5 390 pM, F H 5 37.9 6 1.6%, F M 5 57.7 6 1.6%, in the absence of Gpp(NH)p (F), and F H 5 19.4 6 2.2%, F M 5 76.1 6 2.1%, in the presence of 100 mM Gpp(NH)p (E). D97.103N K M 5 1000 6 80 mM, total [ 3H]NMS 5 35.7 nM, total receptor 5 6.5 nM, in the absence of (F) and presence of 100 mM Gpp(NH)p (E). Fraction of total receptor specifically bound is plotted.

with pilocarpine was less than the other agonists at low receptor density, displaying partial agonism, and equal in extent to the other agonists at high receptor density (Table V). In addition, the effect on adenylyl cyclase activity was biphasic, inhibitory at low agonist concentrations, and stimulatory at high agonist concentrations. This stimulatory phase was not sensitive to pertussis toxin and was only observed at relatively high expression levels and increased proportional to receptor density. The EC 50 for stimulation of adenylyl cyclase was inversely proportional to receptor density. The D97N mutant was the most similar to the wild type of the mutant receptors. Cell lines expressing this mutant inhibited adenylyl cyclase to an extent indistinguishable from that of the wild type and stimulated phospholipase C to an extent slightly less than the wild type at comparable receptor densities. The stimulation of adenylyl cyclase observed in the wild type was also observed in the D97N mutant, but could not be characterized in detail because this mutant did not express at sufficiently high levels. The changes in EC 50 values for agonist stimulation of phospholipase C and inhibition of adenylyl cyclase were consistent with the decreased agonist affinity.

The D69N and D69.97N mutant-expressing cell lines did not stimulate phospholipase C or inhibit adenylyl cyclase activity with the agonists acetylcholine, carbachol, or pilocarpine, but did so with the agonist oxotremorine M (Figs. 2 and 3, Tables IV and V). The changes in EC 50 values for these activities were consistent with the decreased oxotremorine M affinity. Acetylcholine and carbachol partially stimulated adenylyl cyclase activity in the D69N mutant-expressing cell line, but oxotremorine M produced no stimulation. The EC 50 values of this stimulation were similar to those observed with the wild type (26). Pertussis toxin did not eliminate the stimulatory phase of adenylyl cyclase activity, which suggested that it was not a G i- or G odependent process, as reported for the wild type (26). The EC 50 values for the stimulation of adenylyl cyclase in D69N were not significantly different from the wild type but the maximal effects were less than that found in the wild type at comparable receptor density. The D97.103N mutant expressed antagonist binding sites at high enough levels to compare effector coupling properties with those of the wild type and the D97N mutant receptor. At receptor densities at which responses were observed for the wild type and the D97N

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VOGEL ET AL. TABLE II

DDG9° for m2 Muscarinic Receptor Aspartate Mutants

D69N Antagonists NMS 0.094 6 0.043 QNB N.S. AF-DX 116 N.S. Pirenzepine 0.53 6 0.16 Hyoscyamine 20.24 6 0.10 Gallamine 1.21 6 0.13 b Agonists—K H Acetylcholine Carbachol Oxotremorine M Pilocarpine Agonists—K M b Acetylcholine 0.38 6 0.07 Carbachol 0.55 6 0.08 Oxotremorine M 1.28 6 0.29 Pilocarpine N.S. Agonists—K L b Acetylcholine N.S. Carbachol N.S. Oxotremorine M 0.26 6 0.15 Pilocarpine 0.33 6 0.19

D97N

D69.97N

N.S. 1.01 6 0.03 0.69 6 0.11 0.55 6 0.14 0.23 6 0.10 N.S.

0.58 6 0.05 1.12 6 0.10 N.S. 0.89 6 0.14 0.82 6 0.26 0.48 6 0.19

D69.103N

4.46 6 0.05

D97.103N 2.88 6 0.05 3.88 6 0.04 2.60 6 0.14 2.05 6 0.14 2.57 6 0.12 2.10 6 0.11

D69.103N2 D69N a

4.42 6 0.04

D97.103N2 D97N a

D69.97N2 (D69N 1 D97N)

2.84 6 0.04 0.445 6 0.014 2.87 6 0.01 N.S. 1.91 6 0.08 20.64 6 0.15 1.50 6 0.09 20.19 6 0.09 2.34 6 0.06 0.83 6 0.11 2.10 6 0.07 20.73 6 0.11

1.86 6 0.25 0.83 6 0.19 1.08 6 0.16 1.35 6 0.15 1.84 6 0.18 0.54 6 0.24 1.50 6 0.08 0.38 6 0.12

0.99 6 0.23 1.05 6 0.09 1.23 6 0.25 0.34 6 0.22

1.39 6 0.22 0.91 6 0.12 2.0 6 1.3 1.56 6 0.22

0.68 6 0.09 0.96 6 0.06 1.80 6 0.19 0.95 6 0.47

6.57 6 0.26 5.92 6 2.29 6.31 6 0.24 3.31 6 3.28

6.54 6 0.14 5.19 6 0.04 4.77 6 0.07 3.51 6 0.05 5.36 6 0.09 5.47 6 0.20 4.27 6 0.20

6.19 6 0.21 5.4 6 2.0 5.03 6 0.25 3.4 6 3.0

4.70 6 0.16 21.23 6 0.11 4.66 6 0.22 N.S. 3.27 6 0.05 21.55 6 0.17 3.13 6 0.09 N.S. 20.81 6 0.16 4.44 6 0.11 N.S. 3.49 6 0.87 N.S. 2.71 6 0.13 20.94 6 0.22

Note. DDG9° 5 DG9°mutant 2 DG9°wild-type. DDG9° values were calculated from data in Table I and are presented as kcal/mol 6 95% confidence interval (P 5 0.05); N.S., value not significantly different from zero (P , 0.05). a DDG9° for D103N mutation calculated from the difference between the double and single mutants. b Indicates the equilibrium constant for which the DDG9° values were calculated.

mutant, the D97.103N mutant-expressing cell line did not stimulate phospholipase C or inhibit adenylyl cyclase activity at saturating agonist concentrations. TABLE III

Irreversible Thermal Inactivation of Mutant and Wild-Type m2 Muscarinic Receptors in the Presence and Absence of Saturating QNB

Wild type D69N D97N D69.97N D69.103N D97.103N

2QNB

1QNB

DPairwise a

54.1 6 0.4 (15) 56.1 6 1.3 (3) 53.8 6 1.1 (3) 51.0 6 2.8 (4) 52.9 6 1.0 (3) 50.4 6 3.4 (3)

60.2 6 0.3 (16) 62.1 6 0.6 (3) 57.6 6 1.9 (3) 55.4 6 2.4 (4) 53.1 6 0.5 (3) 50.3 6 3.0 (3)

6.2 6 0.2 (15) 6.1 6 1.6 (3) 3.8 6 0.7 (3) 4.6 6 1.3 (4) N.S. (3) N.S. (2)

Note. Comparative T 0.5 values were determined in membrane preparations equilibrated with QNB either before or after a 1-h temperature exposure. T 0.5 is defined as the temperature at which half the QNB binding sites were lost. Mean T 0.5 values (°C) are presented 6 95% confidence (P 5 0.05) interval and the number of experiments is indicated in parentheses. a The stabilizing effect of QNB calculated as the paired difference for experiments that simultaneously determined the T 0.5 in the presence and absence of QNB. N.S., QNB did not significantly affect stability (P , 0.05).

DISCUSSION

Double mutant cycles have been used to study interactions between amino acids that are involved in enzymatic catalysis (30) and stabilization of protein structure (31). We applied this methodology to examine the relationship between three aspartate residues in transmembrane helices 2 and 3 involved in muscarinic receptor agonist binding and signal transduction by changing these residues to asparagine. Changing an aspartate to an asparagine is a conservative mutation designed to disrupt either an intramolecular ionic bond or an ionic bond occurring between the receptor and the ligand amine. These residues are of similar size and hydrophobicity and both are capable of hydrogen bonding, although aspartate can only be an acceptor, while asparagine can be both donor and acceptor. Individually, the substitution of asparagine for aspartate affects agonist binding (D97N), signal transduction (D69N), or both (D103N). Potential site–site interactions were assessed by calculating the value of DDG9° (Table II) using Eq. [5]. For a double mutant cycle in which each of the two mutations are independent (i.e., mutation at either location does not affect the properties of the second mutation) the following relationship should apply:

289

DOUBLE ASPARTATE MUTAGENESIS IN THE m2 MUSCARINIC RECEPTOR TABLE IV

Effect of Aspartate Mutant Muscarinic Receptors on Phospholipase C Stimulation n Wild type b Acetylcholine Carbachol Oxotremorine Pilocarpine D69N Acetylcholine Carbachol Oxotremorine Pilocarpine D97N Acetylcholine Carbachol Oxotremorine Pilocarpine D69.97N Acetylcholine Carbachol Oxotremorine Pilocarpine D97.103N Acetylcholine Carbachol Oxotremorine Pilocarpine

M

M

M

M

M

19 27 34 12 5 5 5 2 4 6 4 2c 3 7 7 2

EC 50 (mM) a 0.13 6 1.41 6 0.45 6 14.1 6

0.03 0.16 0.11 3.5

1.0 6 0.6 23 10.8 4.2 43

17

6 15 6 1.4 6 1.2 6 21

6 5

5 4 5 4

Fold stimulation a

[R] (10 6 receptors/cell)

1.2–2.3 1.2–2.7 1.2–2.4 1–1.8

0.01–1.3 0.07–1.8 0.05–1.8 0.07–1.3

N.S. N.S. 1.41 6 0.06 N.S.

0.6–5.0 2.7–5.0 2.7–5.0 2.7–2.9

6 6 6 6

0.17 0.24 0.13 0.08

0.3–0.4 0.3–1.6 0.3–0.4 0.4

N.S. N.S. 1.42 6 0.05 N.S.

0.4–1.1 0.4–1.1 0.3–1.0 0.6

N.S. N.S. N.S. N.S.

0.2–0.7 0.2–0.7 0.2–0.7 0.2–0.7

2.13 1.88 1.79 1.47

Note. Values for EC 50 and fold maximal stimulation of phospholipase C are shown for the indicated receptor site densities. n, number of experimental determinations; N.S., no significant stimulation was observed. a EC 50 and fold maximal stimulation values are presented 6 standard deviation or for wild-type fold maximal stimulation as a range that increased with [R]. b Wild-type data include experiments where the cell surface receptor density was reduced by incubating the cells with QNB to provide a wide range of receptor densities for comparison with mutants. c In two additional experiments pilocarpine failed to stimulate phospholipase C significantly ([R] 5 0.3– 0.4 3 10 6 receptors/cell).

DDG9°112 5 DDG9°1 1 DDG9°2 .

[6]

If Eq. [6] does not apply, it suggests that the mutation at one site influences the properties of the second site. The free energies of interaction between the sites were assessed from the mutant ligand binding properties, and data from thermal inactivation and effector coupling studies support the conclusion that the three aspartate to asparagine mutations are independent in their effects on the m2 muscarinic receptor properties. In addition, novel results were also found suggesting that the D69N mutant for the m2 muscarinic receptor was still capable of modest signal transduction in a manner suggestive of active conformations that are different from those in the wild-type receptor. Ligand Binding Properties Ligand binding data for the wild-type and mutant m2 muscarinic receptors follow the law of mass action for simple competitive binding equilibria (Eqs. [2]–[4]). Therefore, the effects of a mutation on ligand dissoci-

ation constants (Table I) and the derived free energy differences (Eq. [5], Table II) could be unambiguously compared. Antagonists bind to a single competitive site and affinities were determined from either direct or competition binding experiments. Agonist binding data from competition binding experiments were fit to multiple classes of independent binding sites, where the high affinity site was attributed to the receptor–G protein complex, the intermediate affinity site to the free receptor, and the lowest affinity site to a physically undefined state (26). All mutants, with the exception of D97N, failed to exhibit guanine nucleotide-sensitive high-affinity binding. Therefore, the relative amount of receptor–G protein complex present at equilibrium must be either lower than approximately 5% of the total receptor sites or have affinities that were similar to the free receptor (K H ' K M). Thus, the effect of mutations on agonist affinity for the free receptor was evaluated using the intermediate affinity binding constant, K M (Table I), to calculate DDG9° values (Table II).

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VOGEL ET AL. TABLE V

Effect of Aspartate Mutant Muscarinic Receptors on Adenylyl Cyclase Activity

Wild type c Acetylcholine Carbachol Oxotremorine Pilocarpine D69N Acetylcholine Carbachol Oxotremorine Pilocarpine D97N Acetylcholine Carbachol Oxotremorine Pilocarpine D69.97N Acetylcholine Carbachol Oxotremorine Pilocarpine D97.103N Acetylcholine Carbachol Oxotremorine Pilocarpine

M

M

M

M

M

n

EC 50i (mM) a

Inhibition (%) b

EC 50s (mM) a

Fold stimulation b

[R] (10 6 receptors/cell)

7 18 11 17

0.005–0.093 0.0002–39 0.003–0.45 0.8–60

78.3 6 3.9 63.2 6 2.0 76.0 6 1.4 30–79

1.3–33,000 4.8–180 1.6–10,000

0–1.6 0–2.6 0–1.2 N.S.

0.2–2.1 0.04–2.5 0.2–3.0 0.1–2.2

N.S. N.S. 40.6 6 1.6 N.S.

0.56 6 0.23 d 1.15 6 0.40 d

1.28 6 0.05 1.35 6 0.08 N.S. N.S.

0.8–5.7 0.8–5.7 0.8–5.7 3.5

75.9 6 3.5 75.6 6 3.1 80.3 6 1.5 28–74

10 f 730 f 36 f

0.5 f 0.7 f 0.6 f N.S.

0.6–1.1 0.2–1.1 0.2–1.1 0.2–1.1

N.S. N.S. 41 6 6 N.S.

N.S. N.S. N.S. N.S.

0.2–1.1 0.2–1.1 0.2–1.1 0.5

N.S. N.S. N.S. N.S.

N.S. N.S. N.S. N.S.

0.1–1.4 0.1–1.4 0.1–1.4 0.1–1.0

6 4 10 2 2 5 4 5 7 9 8 2 3 3 5 2

10.2 6 2.8 d 0.13–0.39 e 0.49–10 e 0.1–3 e 19–200 e

167 6 37 g

Note. Values for EC 50, percentage of inhibition, and fold stimulation of adenylyl cyclase are shown for the indicated receptor site densities. n, number of experimental determinations; N.S., no significant effect was observed. a EC 50 values for the inhibitory (EC 50i) and the stimulatory phase (EC 50s) that are reported as a range varied inversely with [R], whereas values that are reported 6 standard deviation appeared to be constant over the indicated range of [R]. b Inhibition and stimulation values that are reported as a range varied proportionally with [R], whereas values that are reported 6 standard deviation appeared to be constant over the indicated range of [R]. c Wild-type data include experiments where the cell surface receptor density was reduced by incubating the cells with QNB to provide a wide range of receptor densities for comparison with mutants. d The observed wild-type oxotremorine M EC 50i for this receptor site density was 7–9 nM; the observed wild-type EC 50s was 1–10 mM for acetylcholine and 4 –20 mM for carbachol. e The observed wild-type EC 50i for [R] 5 1.1 3 10 6 receptors/cell was 9.6 nM, 40 nM, 8.5 nM, and 3 mM for acetylcholine, carbachol, oxotremorine M, and pilocarpine, respectively. f Data were the results of a single experiment at 1.1 3 10 6 receptors/cell; in addition, oxotremorine M stimulated activity at 0.6 3 10 6 receptors/cell (150 mM, 0.3-fold), whereas acetylcholine and carbachol did not. g The observed wild-type EC 50i for [R] 5 1.1 3 10 6 receptors/cell is 8.5 nM.

Agonist and antagonist affinities for the D69N, with the exception of gallamine, pirenzepine, and oxotremorine M, were similar to the wild-type values. The D97N mutation, however, resulted in 3- to 5-fold lower antagonists affinities (with the exception of NMS, hyoscyamine, and gallamine) and 2- to 25-fold lower agonist affinities. The results of the D69N and D97N single mutations were similar to those previously reported for the m1 subtype (9). The double mutant D69.97N showed ligand binding properties that reflected a summation of the two single mutations. The double mutant D69.97N ligand binding properties reflected a summation of the two single mutants. The DDG9° values for D69.97N were within 1 kcal/mol of the DDG9° values summed from D69N and D97N (Table II, column 9).

Thus, it appears that little, if any, interaction occurs between these two sites. Evaluation of double mutant thermodynamic cycles that included the D103N mutation were hampered by low expression and weak ligand binding. The D103N mutation either introduced a barrier to proper protein folding or processing, or receptor expressed on the cell surface was incapable of binding ligands. However, the receptor concentration of the D97.103N-expressing cell line was sufficient to accurately determine ligand dissociation constants. The effect of D97N was apparently compensatory in this double mutant, either increasing the efficiency of expression or stabilizing a state capable of binding ligands, albeit with very low affinity. In addition to data for the D97.103N mutant, limited

DOUBLE ASPARTATE MUTAGENESIS IN THE m2 MUSCARINIC RECEPTOR

291

FIG. 2. Stimulation of phospholipase C by aspartate mutant muscarinic receptors. Illustrated experiments were conducted on attached CHO cells expressing mutant receptor at the following levels (receptors/cell): D69N, 5.0 3 10 6 (E, h) and 2.9 3 10 6 (‚, ƒ); D97N, 0.4 3 10 6; D69.97N, 0.6 3 10 6; and D97.103N, 0.4 3 10 6 (E, h) and 0.7 3 10 6 (‚, ƒ). The lines represent logistic fits of the data (as described under Materials and Methods) where significant stimulation was observed. Symbols: acetylcholine (E), carbachol (h), oxotremorine M (‚), pilocarpine (ƒ); filled symbols indicate response in the presence of the antagonist hyoscyamine.

FIG. 3. Effect of aspartate mutant muscarinic receptors on cAMP formation. Illustrated experiments were conducted on attached CHO cells expressing mutant receptor at the following levels (receptors/cell): D69N, 5.7 3 10 6 (E, h, ‚) and 4.9 3 10 6 (ƒ); D97N, 1.1 3 10 6; D69.97N, 0.8 3 10 6 (E) and 0.7 3 10 6 (h, ‚, ƒ); and D97.103N, 1.4 3 10 6 (E), h, ‚) and 1.0 3 10 6 (ƒ). The lines represent logistic fits of the data (as described under Materials and Methods) where significant stimulation was observed. Fitted parameters for these and other experiments are summarized in Table V. Symbols: acetylcholine (E), carbachol (h), oxotremorine M (‚), pilocarpine (ƒ); filled symbols indicate response in the presence of the antagonist hyoscyamine.

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VOGEL ET AL.

binding data for the D69.103N mutant were available from the CHO cells transfected with pcDNA3 construct. The lack of data for the D103N mutant compromised the analysis to some extent, but the effect of the D103N mutation was observable in the double mutants, and the results were interpreted in terms of possible interactions between Asp103 and the other two aspartates. The DDG9° values for D103N were estimated indirectly by subtraction (Table II, columns 7 and 8): DDG9°D103N 5 DDG9°D69.103N 2 DDG9°D69N 5 DDG9°D97.103N 2 DDG9°D97N. Since the analysis of the D69N, D97N double mutant cycle indicated that the mutations at these sites were additive in nature, the three sites should be additive if the calculated DDG9° values for D103N were the same for both thermodynamic paths. The values were not identical but they were similar. In each case where data were available the calculated DDG9°D103N was greater along the D69N path than along the D97N path by 0.3–1.7 kcal/mol. This difference was considered small, and, thus, the effect of the D103N mutation on ligand binding was considered additive with mutations D69N and D97N. The calculated values obtained for DDG9°D103N, 3– 4 kcal/mol for QNB and 3–5 kcal/mol for agonists, are in the range expected for the loss of an ionic bond (32) and are consistent with this aspartate serving as the counterion for the ligand amine. The strength of this interaction is similar to the well-studied His31–Asp70 salt bridge (3–5 kcal/mol) in T4 lysozyme, which is 75% buried in a cleft near the protein surface and was studied by NMR monitored pK a shifts and site-directed mutagenesis (33, 34). The conclusion that Asp103 is the ligand amine counterion in the m2 muscarinic receptor is supported by amino acid sequence data from the m1 receptor showing that agonist (35) and antagonist (36, 37) affinity alkylating agents modified the homologous aspartate. Thermal Inactivation Studies The change in thermal denaturation behavior of a mutant is often used in studies of protein folding (38) or thermal inactivation of catalytic activity (39) to determine whether the mutation has destabilized the protein structure. In this study, the temperature-dependent irreversible loss of QNB binding activity was measured as an indication of structural stability (Table III). The resulting melting temperature is a kinetic rather than a thermodynamic parameter, and its reduction in a mutant indicates a reduction of the DG ‡ value for inactivation compared to the wild-type receptor. In the absence of QNB, the temperature at which half the receptor population irreversibly lost antagonist binding activity was not significantly different for mutant receptors compared to the wild type. The simplest interpretation of these results is that D69N,

D97N, and D103N mutations did not greatly disturb the structure of the unliganded receptor; however, perturbations not reflected by changes in thermal stability cannot be discounted. Saturating concentrations of QNB stabilized the wild-type receptor to thermal inactivation. This stabilizing effect was lost in the two double mutants that included D103N and was reduced slightly by the inclusion of D97N alone or in the double mutant D69.97N. In the receptor–QNB complex the ligand amine charge is presumably buried within the hydrophobic core of the receptor. Since an unpaired charge is energetically too expensive to bury (32), it is likely paired with an anionic amino acid side chain. The reduced thermal stability of the receptor–QNB complex in the D103Ncontaining mutants can be explained if Asp103 is the amine ligand counterion, as suggested by the ligand binding results. The consequence of neutralizing half of an ion pair between receptor and ligand is the presence of an unpaired charge in the receptor core that destabilizes the structure of the liganded receptor. Since QNB stabilized wild-type and D69N equally, the small reduction in thermal stability observed equally in D97N and D69.97N support the conclusion that the effects of the D69N and D97N mutations are additive. Effector Coupling Properties In CHO cells the wild-type m2 muscarinic receptor couples to the inhibition of adenylyl cyclase and, more weakly, to the stimulation of phospholipase C through pertussis toxin-sensitive G proteins. At very high levels of agonist a robust G protein-mediated stimulation of adenylyl cyclase activity occurs that is not affected by pertussis toxin or dependent on intracellular calcium (26). In agreement with previous studies of the homologous aspartate in the m1 receptor (9), the D97N mutation still couples to these responses (Figs. 2 and 3, Tables IV and V); however, the dose–response curves are right-shifted, consistent with the weaker agonist binding. The results of the D69N mutation for the m2 receptor differed from those previously reported for the m1 subtype, where the mutation eliminated signal transduction (9). In the D69N mutant m2 subtype signal transduction was agonist specific. Acetylcholine, carbachol, and the partial agonist pilocarpine failed to stimulate phospholipase C or inhibit adenylyl cyclase, but oxotremorine M still permitted weak but clearly observable coupling to these effector systems. Similar results were also found for the D69.97N double mutant, and the EC 50 values for oxotremorine M were right-shifted, consistent with the weaker agonist binding. The rightward shift in EC 50 values was especially true for adenylyl cyclase inhibition (Table V), which is correlated with the absence of observable guanine nu-

DOUBLE ASPARTATE MUTAGENESIS IN THE m2 MUSCARINIC RECEPTOR

cleotide sensitive binding site in these mutants (K H values, Table I). However, acetylcholine and carbachol but not oxotremorine M promoted adenylyl cyclase stimulation in D69N with EC 50 values similar to those of wild-type m2 receptors. Acetylcholine and carbachol were ineffective in causing adenylyl cyclase stimulation in the D69.97N mutant. However, these results are inconclusive because the mutant was not expressed at a sufficient level to observe this response. Adenylyl cyclase stimulation was strongly dependent on expression levels (26) and was only unambiguously found in wild-type and the D69N mutant-expressing cell lines. These results suggest that D69N-containing mutants have one or more agonist states that differ from that found in the wild-type receptor. The wild type couples strongly to G proteins, showing a significant fraction of receptor in the high-affinity, guanine nucleotide-dependent, agonist binding state. The D69N mutant does not form sufficient quantities of this state to be observable with the agonists tested. Acetylcholine and carbachol apparently do not activate a sufficient number of G proteins to couple to either adenylyl cyclase inhibition or phospholipase C stimulation. Oxotremorine M, however, must induce a unique conformation such that enough G proteins can be activated to cause weak coupling to these responses even though high-affinity, guanine nucleotide-dependent, agonist binding remains at undetectable levels. The agonists acetylcholine and carbachol do, however, induce a conformation in the D69N mutant capable of activating adenylyl cyclase, a response that is independent of pertussis toxin-sensitive G proteins in CHO cells. This state is not found for the agonist oxotremorine M. This pattern of agonist-specific activity suggests that the two conformational states are distinct in the D69N mutant. If the D69N mutation affected only receptor–G protein interactions then alterations in effector coupling should apply uniformly for all agonists rather than in an agonist-specific manner. At this time, it is unknown whether this observation applies to wild-type m2 receptors since acetylcholine, carbachol, and oxotremorine M all couple effectively to adenylyl cyclase inhibition, phospholipase C stimulation, and adenylyl cyclase stimulation in CHO cells. Agonists that selectively activate muscarinic subtypes are being sought as potential therapeutics. The selective conformational activation observed in the D69N mutant suggest that it may be possible to design drugs that cause similar behavior for wild-type receptors and affect a finer level of control of muscarinic activity than subtype specific activation. Similar results were reported for the mutation of the homologous aspartate in the a 2-adrenergic receptor where coupling to activation of inwardly rectifying potassium channels was lost but coupling to adenylyl cyclase inhibition and

293

voltage-dependent calcium channels was maintained (40). The effect of the D69N mutation was to reduce the level and alter the agonist specificity of signal transduction activity, whereas the only effect of the D97N mutation was to reduce agonist affinities. The D69.97N mutant had the same altered pattern of signal transduction activity as D69N in addition to lower agonist affinities. These results support the conclusion that the effects of D69N and D97N are additive. The effects of the D103N mutation on effector coupling were difficult to determine due to the low binding activity found in the D103N-containing cell lines. Because the D97.103N cell line expressed a similar number of antagonist binding sites per cell as the D97N mutant, it was possible to evaluate the contribution of the D103N mutation by comparing the two cell lines. The D97.103N double mutant did not show either highaffinity guanine nucleotide-sensitive agonist binding or the ability to couple to any effector system (Tables IV and V). Since the D97N mutation affects only ligand binding properties, these results indicate that Asp103 has an essential role in m2 muscarinic receptor signal transduction in addition to its function in ligand binding. The homologous mutation in the m1 muscarinic receptor eliminated acetylcholine-promoted phospholipase C stimulation, whereas the glutamate mutation preserved partial activity (41). Differing effects of mutating the aspartates homologous to Asp103 in the m2 muscarinic receptor to asparagine have been found in other G protein-coupled receptors that bind biogenic amines. In the a 2- (13) and b 2-adrenergic (11), 5-hydroxytrypamine A1 (14), and 5-hydroxytrypamine 2 (15) receptors the effect of this mutation appears limited to ligand affinity. The signal transduction activity of these receptors was preserved, whereas signal transduction was eliminated in the m1 (41) and m2 muscarinic receptors. The D103N mutant receptor was poorly expressed on the cell surface, but the homologous mutations in b 2-adrenergic (42), a 2Aadrenergic (13), 5-hydroxytrypamine 2 (15), and histamine H 1 (43) receptors were well tolerated. Asparagine mutations at this homologous site in the histamine H 2 (44) and the dopamine D 2 (45) receptors also resulted in undetectable cell surface expression; however, it is unclear if this was due to poor protein expression or dramatically decreased ligand affinity. In conclusion, the method of double mutant cycles was used to examine whether mutations of any of the three transmembrane aspartates (Asp69, Asp97, and Asp103) to asparagine affects the properties of the amino acids at the remaining two positions. The analysis of these cycles suggests that the mutant effects on the free energy of ligand binding, signal transduction, and thermal stability were, to a large degree, independent in nature, and that structural perturbations are

294

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not propagated between these residues in a functionally relevant manner. Ligand binding and thermal inactivation results both agreed with previous work (41) indicating that Asp103 is the ligand amine counterion, and that this residue is important for the maintenance of active receptor conformation(s) required for signal transduction. The signal transduction activity of the D69N mutant, unlike wild-type, is strongly agonist dependent. This observation is explained by suggesting that this mutant can assume active conformations that differ from the wild-type m2 muscarinic receptor. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants HL23632 and ES00210.

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