structural studies on d2 dopamine receptors: mutation of a histidine residue specifically affects...

Journal of Neurochemistry Raven Press, Ltd., New York 0 1994 International Society for Neurochemistry

Structural Studies on D, Dopamine Receptors: Mutation of a Histidine Residue Specifically Affects the Binding of a

Subgroup of Substituted Benzamide Drugs

Robert Woodward, Sarah J. Daniell, Philip G. Strange, and Louise H. Naylor

Biological Laboratory, The University, Canterbury, Kent, England

Abstract: A histidine residue (His394) that is likely to be located in the ligand-binding region of the D, dopamine receptor has been mutated to a leucine (Leu394), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na+/H+ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the

residue with these compounds. Key Words: D, dopamine receptor-Mutagenesis-Ligand binding-Histi- dine residue-Substituted benzamides. J. Neurochem. 62, 1664-1 669 (1 994).

It is now clear from biochemical and pharmacologi- cal studies and more recently molecular biological studies that a family of dopamine receptors exists (D1, D,, D3, D,, and D,) that have different pharmacologi- cal properties and different cellular localizations (Sib- ley and Monsma, 1992; Civelli et al., 1993). Under- standing the basis of the specificity of these receptor subtypes and how they bind ligands will be very important in guiding selective drug design. The D, subtype is of particular interest as it is a major receptor for dopamine in the brain and is also a target for anti- parkinsonian and antipsychotic drugs. We are analys- ing this particular subtype in detail to understand the determinants of ligand binding.

Chemical modification and pH dependence studies of ligand binding have implicated a carboxyl group in the binding of antagonists such as spiperone to D, dopamine receptors (Williamson and Strange, 1 990). This carboxyl group may correspond to Asp114 in the third putative transmembrane spanning region of the receptor (Bunzow et al., 1988). Analogous aspartic

acid residues are found in equivalent positions in all G protein-linked receptors that bind cationic amines and are thought to participate in an electrostatic interaction between ligand and receptor (Strader et al., 1989). Mutagenesis studies of Asp'14 support this idea (Mansour et al., 1992). Studies of pH dependence of ligand binding have also implicated another group having a pKa of -7 that specifically affects the binding of substituted benzamide drugs such as (-)-sulpiride to D, receptors (Williamson and Strange, 1990; Presland and Strange, 199 1). Molecular modelling of the D, dopamine receptor has identified a histidine residue (His394 in the long form of the D, receptor) about one-third of the way down the sixth putative transmembrane domain of the receptor (Livingstone et al., 1992). This histidine residue is therefore likely to be within the ligand-binding cavity, in the region where ligands are thought to bind (Tota et al., 199 l), and could provide the group of pKa 7. In this study, therefore, we have performed site-directed mutagenesis studies on the rat D2(,ong) receptor to investigate the role of this histidine residue in the binding of a range of antagonist drugs.

MATERIALS AND METHODS

Mutagenesis and expression Mutant rat DZ(,ong) receptors were prepared using the oligo-

nucleotide-directed mutagenesis system supplied by Amer- sham International. The oligonucleotide primer, 23 bases in length, was made to contain a single mismatch in the mid- dle ofthe sequence so as to change the codon CAC (His) to a CTC (Leu). The rat Dz(,ong) cDNA contained in the pBlue- script vector (Stratagene) was obtained from Philippe Ver- nier and Jacques Mallet (CNRS, Gif sur Yvette, France). This was used to make single-stranded DNA for the mutagenesis reaction. Positive clones were identified using di-

Received August 2, 1993; revised manuscript received Sep- tember 7, 1993; accepted September 7, 1993.

Address correspondence and reprint requests to Dr. P. G. Strange at Biological Laboratory, The University, Canterbury, Kent, CT2 7NJ, U.K.

1664

D2 DOPAMINE RECEPTOR MUTAGENESIS 1665

deoxy seque cing (Sanger et al., 1977). A BglII/BfrI frag-

mammalian expression vector pSVL, which already contained the D, receptor cDNA, exchanging the native BglII/ BfrI fragment for the mutant fragment. The cloning sites were then sequenced to confirm the fidelity of the new con- struct. Mutant pSVL D, Leu and native pSVL D, His plasmids were used for subsequent transient transfections in COS-7 cells. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, 2 mM glutamine, and 1 mM sodium pyru- vate. Two days before transfection cells were subdivided at 1 X lo6 cells/ 1 75-cm2 flask, and the diethylaminoethyl-dextran method was then used to transfect cells with 50 pg of the appropriate plasmid. The transfection method used was similar to that described by Gorman (1985) but included a 30-min incubation of the cells at 37°C in an atmosphere of 5% CO, with Dulbecco's modified Eagle's medium containing 0.5 mg/ml of diethylaminoethyl-dextran and plasmid DNA. Chloroquine (100 p M ) was then added, and the cells were incubated for a further 2.5 h. The transfection mixture was aspirated, and the cells were shocked for 2 min with 10% dimethyl sulphoxide before addition of complete medium and incubation at 37°C in an atmosphere of 5% C02 for 72 h.

Preparation of cell membranes Medium was removed, and the cells were washed with

buffer containing 20 mM HEPES (pH 7.4), 6 mM MgCI,, 1 mM EDTA, 1 mM EGTA, and 0.25 M sucrose at 4°C. The wash buffer was replaced with the same buffer, and the cells were scraped from the flask and homogenized with 30 strokes of a Dounce homogenizer. The homogenate was centrifuged at 1,700 g for 10 min at 4"C, and the superna- tant was centrifuged at 48,000 g for l h at 4°C. The resulting pellet was resuspended in buffer containing 20 mM HEPES (pH 7.4), 6 mM MgCI,, 1 mMEDTA, and 1 mMEGTA at 4°C and stored in 1.5-ml aliquots at -80°C.

Ligand-binding assays For ligand-binding assays 30 p g of membrane prepara-

tion was incubated in triplicate with [3H]spiperone (Amer- sham International; specific activity, 28 Ci/mmol; 0.25 nM for competition assays or 15 pM-1 nM for saturation analysis). Competing drug was added at the appropriate concen- tration, and the reaction mixture was made up to 1 ml with buffer [20 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM EGTA, and 120 mM NaCI]. Specific binding was determined as the binding that was inhibited by 3 pM (+)-butaclamol. Reactions were incubated at 25°C for 45 min before harvesting by filtration through GF/B glass fibre filters on a Brandel cell harvester. Filters were immediately washed with three 4-ml washes of phosphate-buffered saline (0.14 M NaCl, 3 mM KCI, 1.5 mM KH,P04, and 5 mM Na2HP04, pH 7.4) at 4°C. The filters were then soaked in 2 ml of LKB Optiphase Hisafe 3 scintillation fluid for 6 h before determination of bound radioactivity by liquid scintillation counting. The following substances were generous gifts that we gratefully acknowledge: haloperidol from Jans- sen; flupenthixol from Lundbeck; DO 710 from Dr. A. Mann (CNRS, Strasbourg, France); raclopride and remoxipride from Astra; nemonapride (YM 09151-2) from Ya- manouchi; clebopride from Almirall; amisulpride, LUR 2366, sultopride, and tiapride from Synthelabo, sulpiride from Delagrange, and benzamide- 1 and metoclopramide

ment contain 7 ng the point mutation was subcloned into the 1750

0

1500

1250

1000

750

500

250

0.00 0.20 0.40 0.60 0.80 1.00

[%]spiperone (nM)

FIG. 1. Saturation analysis of [3H]spiperone binding to native (0) and Le~~'~-mutant (0) D, dopamine receptors expressed in COS- 7 cells. Saturation binding analysis was carried out as described in Materials and Methods. The data are from a single experiment replicated as in Table 1, and the data fit best to a one-binding-site model in both cases: native, B,,, = 1,627 fmol/mg, K, = 0.044 nM; mutant, B,, = 1,065 fmol/mg, K, = 0.056 nM.

from SmithKline Beecham. Other substances were obtained from commercial suppliers.

The binding data were analysed using the EBDA and LI- GAND programmes (McPherson, 1985) to determine KD or Ki values. Student's t test was used to determine the signifi- cance of any difference in Ki/KD affinity between native and mutant receptors.

RESULTS

COS-7 cells were transfected with either the plasmid containing the native D, receptor (pSVLD2 His) or one in which the His364 codon had been mutated to Leu394 (pSVLD2 Leu), and cell membranes were prepared 72 h after transfection. [3H]Spiperone binding was detected in cells transfected with either of these plasmids and showed saturable binding levels of - 1,000 fmol/mg of membrane protein (Fig. 1). Ex- pression levels were similar for both plasmids, and the saturation data for [3H]spiperone binding were consistent with a one-site binding model. In addition, membrane preparations from untransfected cells gave no detectable levels of specific [3H]spiperone binding (data not shown). Both native and mutant cell membrane preparations showed a similar high affinity for binding [3H]spiperone (KD of -40 pM, Fig. 1 and Table 1).

The effects of the mutation were then tested using other antagonist drugs in competition assays with [3H]spiperone (Table 1 and Fig. 2). In all cases the competition data fitted to a one-binding-site model. The data show that for a group of antagonists of different chemical classes [the butyrophenone haloperidol, the thioxanthene cis-flupenthixol, and the stereoisomeric antagonist (+)-butaclamol] the affinity

J . Neurochem.. Vol. 62, No. 5, 1994

1666 R. WOODWARD ET AL.

TABLE 1. Effect of Led94 mutation on antagonist binding at D, dopamine receptors

Antagonist

(+)-Butaclamol cis-Flupenthixol Haloperidol Spiperone

Amisulpride Benzamide- 1 Clebopride DO 710 Nemonapride LUR 2366 Metoclopramide Raclopride Remoxipride ( -)-Sulpiride Sultopride Tiapride

Ki or K D (nM)

Native Leu mutant

3.6 f 1.1 2.15 f 0.25 2.6 f 0.7 1.24 f 0.35 1.2 f 0.2 0.76 f 0.14

0.032 f 0.01 0.048 f 0.0 I

9.4 f 0.8 25.8 f 5.6 1.74 f 0.04 0.18 f 0.03 0.94 f 0.22 0.15 f 0.04

4.7 f 1.6 60.8 f 33 0.2 1 f 0.09 0.13 f 0.04

1.6 f 0.1 6.2 f 0.7 46 f 4 2.1 -t 0.6 3.5 f 0.4 5.9 f 1.6 329 f 0.5 566 f 133 14.7 f 3.1 116.2 f 34.4 3.3 f 1.3 66.6 f 15.7 114 f 15.7 41 1.5 f 42.3

Ratio (mutant/native)

0.59 0.48 0.63 1.5

2.7" 0.10" 0.15"

12.9" 0.6 1 4" 0.04" 1.7 1.7 7.9"

4.0" 20.24

The binding of a range of antagonist drugs to native and Leu394 mutant D, dopamine receptors was determined as described in Ma- terials and Methods using [3H]spiperone binding in saturation or competition analyses. KJK, values were determined by computer nonlinear least squares fitting (EBDA/LIGAND) to single-binding site models, which provided the best fit to the data in all cases. Data are mean f SD values for three or more experiments.

p < 0.05 by t test for difference in affinity between native and Leu-mutant receptors.

of binding to the native and Leu394 mutant receptor membranes was very similar. When substituted benzamide antagonists were tested, some members of this class of compound displayed similar affinities for both the native and the mutant receptors, e.g., raclopride, remoxipride, and nemonapride. However, certain members of this class of drug did show significant differences in affinity for the Ledg4 mutant relative to the native receptor (Table 1). The affinity at the Leu394 mutant was lower relative to the native receptor for some compounds, e.g., for sultopride, (-)-sulpiride, and DO 7 10 the affinity was eight- to 20-fold lower, whereas the affinity was increased for certain drugs, e.g., for benzamide- 1, metoclopramide, and clebopride the affinity was up to 20-fold higher.

The binding of substituted benzamide drugs to D, receptors is sensitive to the effects of changes in Na+ and H'. Therefore, the sensitivity to the effects of changes in Na' or H+ of the binding of (-)-sulpiride to native and Leu394 mutant receptors was examined. The results showed that the Leu394 mutation did not reduce the Na+ or H+ sensitivity of the binding of sulpiride to the receptor (Fig. 3). In fact, the Leu394 mutant receptor showed a greater sensitivity to changes in these cations.

DISCUSSION In this report we have probed the ligand-binding

site of the D, dopamine receptor by mutating a histi-

dine residue (His394) in the sixth transmembrane domain of this receptor, which is thought to be at the binding site (Livingstone et al., 1992). His394 was mutated to Leu394 to eliminate potential hydrogen bonds or electrostatic bonds to ligands, while retaining the bulk of this residue. The results show that this amino acid residue may be important for the binding of one class of drugs to the receptor, the substituted benzamides.

The properties of this Leu394 mutant receptor were evaluated by expression of mutant DNA in COS-7 cells followed by ligand binding with [3H]spiperone. The affinities of a range of antagonists for the native receptor expressed in COS-7 cells are strikingly similar to those seen for the receptor expressed in Chinese hamster ovary cells (Castro and Strange, 1993) or in rat brain (Leonard et al., 1988). Therefore, expression in COS-7 cells produces a receptor with authentic properties. Because the affinities of a wide range of antagonists representative of different chemical classes are largely unchanged by the mutation, it does not appear to affect either the targetting of receptor to the membrane or its gross conformation and folding.

The effects of the mutation are in fact highly specific, affecting significantly only the affinities of certain members of the substituted benzamide class of antagonists. For some of these, e.g., sultopride, sulpiride, tiapride, and DO 7 10, there is a reduction in affinity for the Leu394 mutant receptor, whereas for others, e.g., benzamide- 1, clebopride, and metoclopramide, the affinity increases. The specificity of the mutation is underlined by the fact that the binding of

2 60 -

$ 50 -

to 40 -

7 - - 30 s

20

Cl a .-

-

j o t 0- -13-12-11-10 -9 -8 -7 -6 -5 -4

log [competitor] (MI

FIG. 2. Binding of different ligands to native and L e ~ ~ ~ - m u t a n t D, dopamine receptors. Competition studies with nemonapride (circles), metoclopramide (triangles), and (-)-sulpiride (squares) were performed versus [3H]spiperone binding as described in Ma- terials and Methods to membranes of COS-7 cells expressing native (solid symbols) or mutant (open symbols) receptor. The data are from single experiments replicated as in Table 1 and fit best to one-binding-site models.

J. Neurochem., Vol. 62, No. 5, 1994

D, DOPAMINE RECEPTOR MUTAGENESIS 1667

-9 -8 -7 -6 -5 -4 -3

log [competitor] (M)

100

90

B

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

log [competitor] (M)

FIG. 3. Binding of (-)-sulpiride to native and Leu3=-rnutant D, dopamine receptors and effect of removal of Na+ and of pH change to pH 6. (-)-S~lpiride/[~H]spiperone competition studies were performed as described in Materials and Methods. A Native receptors/Na+ (A), native receptor/no Na+ (A), Leu394 mutant/Na+ (O), and Leu394 mutant/no Na+ (m). B: Native receptor/pH 7.5 (A), native receptors/pH 6.0 (A), Leu3= mutant/pH 7.5 (a), and Leu3= mutant/pH 6.0. For experiments examining the effect of removal of Na+ (A), the Na+ was replaced by N-methyl-o-glucamine HCI. For experiments examining the effects of pH changes (B), the assays were performed in the buffer described in Williamson and Strange (1990). The data are from single experiments replicated as below and fit best to a one-binding-site model. The mean ? SD K, (nM) values for three experiments were as follows: A (effect of changes in Na+), + Na+ versus no Na+, 17 2 7 versus 89 & 2 for the native receptor and 11 1 -t 50 versus 860 It 40 for the Leu3= mutant; B (effect of pH change), pH 7.5 versus pH 6.0, 14 f 1 versus 428 f 23 for the native receptor and 172 f 22 versus 13,800 k 1,950 for the Leu3s4 mutant.

certain substituted benzamides is unaffected by this mutation. Although the effects of the mutation appear to be confined to the substituted benzamide class of antagonists and there are no effects on the binding of the butyrophenone or thioxanthene antagonists tested, it cannot be concluded from the present data that all members of these latter classes of drug will be unaffected.

The binding of the substituted benzamide drugs to the D, dopamine receptor is particularly sensitive to Na+ (Theodorou et al., 1980) and H+ (Williamson

and Strange, 1990), and this could be related to His394. Therefore, the effects of changes in Na+ and H+ on the affinity of sulpiride for the native and Leu394 mutant receptors were tested. The mutation did not reduce the sensitivity of the receptor to either cation so that His394 cannot be responsible for the sensitivity to Na+ or H+. This is also consistent with the rather specific effect of the mutation on certain substituted benzamide drugs as compared with the general sensitivity of these drugs to the effects of Na+ and H+. The substituted benzamide drugs also show differential affinities for the splice variants of the D, receptor, D2(shon) and D2(long) (Castro and Strange, 1993). Here there is a ten- dency for compounds that are affected by the Leu394 mutation to show a larger affinity difference between DZ(shon) and DT(long), but it is not clear how this relates to any potential conformational difference between the two receptor forms.

Thermodynamic studies of the binding of ligands, including substituted benzamides to D, receptors, have divided these compounds into classes whose binding is driven by a change in enthalpy or a change in entropy (Kilpatrick et al., 1986). It is notable that the binding of substituted benzamides affected by the Leu394 mutation is enthalpy driven, whereas the binding of compounds unaffected by the mutation is entropy driven. Again it is difficult to interpret this divi- sion, but it perhaps emphasizes the different modes of binding of different compounds to the receptor.

Some speculations can be made, however, regard- ing the interactions between receptor and ligands that might give rise to the effects seen with the mutation of His394. Because the effects are highly specific, affecting the binding of certain substituted benzamide drugs only, it is unlikely that the effects of the mutation are associated with a conformational change in the receptor. Similarly, it is unlikely that aromatic/aromatic interactions between His394 and the substituted benzamide drugs are being disrupted; otherwise, effects on all the drugs of this type would be expected. The specificity of the effects suggests disruption of specific interactions, and the size of the effects seen is most consistent with the breakage of the hydrogen bond, with much greater effects being expected with the breakage of an electrostatic linkage (Fersht et al., 1985).

To understand the effects further it is necessary to consider the structures of the substituted benzamide compounds tested (Table 2). These fall into four groups, according to the nature of the amide substituent: group I, ( l-ethylpyrrolidin-2-yl)methyl, e.g., sulpiride; group 11, diethylaminoethyl, e.g., metoclopramide; group 111, 1 -benzylpiperidin-4-y1, e.g., clebopride; and group IV, 1 -benzyl-2-methylpyrrolidin- 3-yl, e.g., nemonapride. Dividing the compounds into groups in this way is useful, as it emphasizes structural similarities and differences between the compounds. Also, within a group of structurally related drugs, e.g., group I, the effects of the mutation can then be com-

J. Neurochem., Vol. 62, No. 5. 1994

1668 R. WOODWARD ET AL.

TABLE 2. Structures of substituted benzamide drugs and effects of the Leu394 mutation on their affinity

Fold change

in R, R4 R5 R6 affinity

Group I:

Amisulpride Sulpiride Sultopride DO 710 Raclopride Remoxipride Benzamide- 1 LUR 2366"

H NH2 H H H H H H C1 H H H H NH2 H NH2

Group 11:

0

SOZEt SOZNHZ SOZEt S02NHMe c1 Br c1 SO,Et

H 2.71 H 7.91 H 20.21 H 12.91 OH 1.71 OMe 1.71 H 9.9t H 41

Tiapride H S02Me Metoclopramide NH2 C1

Group 111:

Clebopride

Group IV:

0

41 22t

6t

c ' W i P - C H Z P ~

MeNH OMe CH, Nemonapride 1.6t

LUR 2366 has an N-cyclopropylmethyl substituent.

pared for compounds differing by single functional groups to make mechanistic deductions.

For group I, the drugs that show a reduced affinity for the mutant receptor possess a sulphonamide or sulphone substituent at the 5-position on the benzamide ring, e.g., DO710, sulpiride, and sultopride. If this substituent is replaced by a halide substituent, there is no significant effect of the mutation, e.g., raclopride and remoxipride, although there are other changes in substituents in these compounds. If the 5-sulphone/sulphonamide substituent is replaced by

a chloro substituent and a 4-amino substituent is added (benzamide- 1 ), then an increase in affinity is seen in the mutant receptor. If a 4-amino substituent is present together with a 5-sulphone, the effect of the mutation is reduced compared with the affinity of drugs with the 5-sulphone alone (compare sultopride and amisulpride or LUR 2366) or the 4-amino 5- chloro substituent pattern (compare benzamide- 1 and amisulpride). For group I1 compounds, tiapride has a 5-sulphone substituent, and a decrease in affinity is seen for the mutant receptor, whereas for metoclopramide, with a 4-amino, 5-chloro substitution pattern, a large increase in affinity is seen.

From the behaviour of these two groups of compounds some conclusions may be drawn about how these ligands might bind to the receptor. A 5-sulphone/sulphonamide, if present, leads to a reduction in affinity of the mutant receptors for the compound. A hydrogen bond between His394 and the 5-sulphone/ sulphonamide seems likely, therefore, and this may be in the form of N+-H-'-O-S. El Teyar et al. ( 1988) have proposed, based on structure-activity studies of antagonist binding at Dz dopamine receptors, that for compounds of the substituted benzamide class there is a productive interaction between substituents in the 5-position and a group on the receptor bearing a par- tial positive charge. This suggestion is consistent with the findings here. The mutant receptor shows an increased affinity for the compounds with a 4-amino 5-chloro substituent pattern (benzamide- 1, metoclopramide). One interpretation of these findings is that a 5-chloro substituent interacts unfavourably with the His394 residue, and this unfavourable interaction is relieved by the mutation. This interpretation empha- sises interaction [productive (sulphone/sulphonamide)/unproductive (chloro)] between His394 and the 5-position of the benzamide ring. Arguing against this idea are the nonsignificant effects of the mutation on the affinity of raclopride for the receptor. Raclopride possesses a 5-chloro substituent, but there are also substituent changes elsewhere in the benzamide ring.

An alternative interpretation is that the 5-chloro substitution is neutral with respect to the mutation, but there is an unfavourable interaction between the 4-amino substituent and that is relieved by the mutation. Data consistent with this interpretation are the reduced effects of the mutation on compounds containing 5-sulphone and 4-amino substituents. Also consistent with an unfavourable interaction between the 4-amino substituent and the receptor is the lower affinity of amisulpride for the native receptor compared with sultopride.

Clebopride and nemonapride are single representa- tives of quite different chemical classes and are notable for their high affinities for the D, receptor. This could be related to the presence of a benzyl substituent on the amino group, suggesting a further potential interaction between ligands and the receptor. The effect of the Leu394 mutation is to increase the affinity

J. Neurochem.. Vol. 62, No. 5 , 1994

D2 DOPAMINE RECEPTOR MUTAGENESIS 1669

for clebopride, and this is consistent with the presence of the 4-amino 5-chloro substituent pattern (see above). Nemonapride has a 4-methylamino substituent, but the mutant receptor has an affinity for nemonapride similar to that of the native receptor. The amide substituent of nemonapride is markedly different from that of the other groups of compounds so that its mode of binding to the receptor may be quite different.

This study has therefore uncovered a potential interaction between certain substituted benzamide drugs and the D, dopamine receptor. These drugs probably also interact with the receptor via an electrostatic interaction between their protonated amino group and in the third membrane spanning a-helix. Other interactions, however, remain to be dis- covered to account for the binding affinities. These may include hydrogen bond interactions to serine residues (Cox et al., 1992; Mansour et al., 1992).

In summary, therefore, this study has identified a histidine residue ( His394) in the sixth putative transmembrane region of the D, receptor that may interact with certain substituted benzamide drugs and that may contribute to their binding energies.

Acknowledgment: We thank the Wellcome Trust and the MRC for their financial support, Sue Davies for preparing the manuscript, Karen Cleverly for performing preliminary studies on the Na+/H+ dependence of sulpiride binding, and Frank Brown (SmithKline Beecham) for help in supplying compounds.

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J. Neurochem., Vol. 62, No. 5, 1994

structural studies on d2 dopamine receptors: mutation of a histidine residue specifically affects...

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