Lett. 23, 167-173 25 Nagy, F. and Dickinson, P. S. (1983) J. Exp. Biol. 105, 33-58 26 Dickinson, P. S. and Nagy, F. (1983) J. Exp. Biol. 105, 59-82 27 Nagy, F., Dickinson, P. S. and Moulins, M. (1988) J. Neurosci. 8, 2875-2886 28 Dickinson, P. S., Nagy, F. and Moulins, M. (1988) J. Exp. Biol. 136, 53-87 29 Nusbaum, M. P. and Marder, E. (1989) J. Neurosci. 9, 1591-1599 30 Nusbaum, M. P. and Marder, E. (1989) J. Neurosci. 9, 1600-1607 31 Nusbaum, M. P., Cournil, I., Golowasch, J. and Marder, E. (1989) Soc. Neurosci. Abstr. 15, 366 32 Cournil, I., Meyrand, P. and Moulins, M. (1989) Soc. Neurosci. Abstr. 15, 366 33 Rezer, E. and Moulins, M. (1983) J. Comp. Physiol. 153, 17-28 34 Heinzel, H-G. (1988) J. Neurophysiol. 59, 528-550 35 Heinzel, H-G. (1988) J. Neurophysiol. 59, 551-565 36 Hooper, S. L. and Moulins, M. (1989) Science 244,

1587-1589 37 Nusbaum, M. P. and Marder, E. (1988) J. Exp. BioL 145, 165-181 38 Dickinson, P. S. and Marder, E. (1989) J. Neurophysiol. 61, 833~844 39 Dickinson, P. S., Mecsas, C. and Marder, E. (1990) Nature 344, 155-158 40 Weiss, K. R., Koch, V. T., Koester, J., Mandelbaum, D. E. and Kupfermann, I. (1981) in Neurobiolog~/ of Invertebrates (Slanki, J., ed.), pp. 305-344, Pergamon Press 41 Chiel, H. J., Weiss, K. R. and Kupfermann, I. (1986) J. Neurosci. 6, 2427-2450 42 Mackey, S. L., Kandel, E. R. and Hawkins, R. D. (1989) J. Neurosci. 9, 4227-4235 43 Weiss, K. R., Chiel, H. L. and Kupfermann, I. (1986) J. Neurosci. 6, 2403-2415 44 Chiel, H. J., Weiss, K. R. and Kupfermann, I. (1990) Trends Neurosci. 13, 223-227 45 Frost, W. N. and Getting, P. A. (1989) Soc. Neurosci. Abstr. 15, 1118

Aspects of the structure of the D2 dopaminereceptor P h i l i p G. S t r a n g e

Significant new information on the 1)2 dopamine receptor has recently become available from a combination of protein chemical and molecular genetic analyses. Molecular genetic studies have shown the receptor to be a member of the family of receptors that are linked to G proteins and that have structures predicted to contain seven transmembrane domains. Two distinct species of 1)2 dopamine receptor have beenfound which may differ in their coupling to G proteins; their distributions have been mapped at the nucleic acid level. The l)2 dopamine receptor has been purified from brain and anterior pituitary and characterized. Chemical modification of the brain receptorprovides evidence for the importance of a carboxyl group that interacts with ligands at the receptor binding site. Here, Philip Strange discusses these points and proposes models of receptor-ligand interaction based on the conservation of several aspartic acid residues in receptors that bind cationic amines. It is now well established that the actions of the neurotransmitter dopamine may be accounted for through its interaction with pharmacologically distinct D1 and De dopamine receptors. Several important actions of dopamine, including the central control of motor function and certain behaviours, the inhibition of pituitary hormone secretion and the modulation of cardiovascular function involve D~ dopamine receptors; in addition, they are key sites of action of antiparkinsonian and anti-schizophrenic drugs. Understanding of the mechanism of action of the D2 dopamine receptor is increasing: the receptor interacts with one or more G proteins (members of the Gi/Go family) to inhibit adenylate cyclase or open potassium (K +) channels and possibly to attenuate calcium (Ca2+) channel opening (reviewed in Ref. 1). The precise nature of the G proteins involved is not clear, neither is it clear whether a multiplicity of mechanisms of receptor action predicates multiple pharmacologically distinct receptor subtypes. Until recently, knowledge about the structure of the receptor has been sparse compared with that about TINS, Vol. 13, No. 9, 1990

the structure of other receptors 2. This review summarizes some of the recent major advances in the structural analysis of the D2 dopamine receptor. Purification of D2 dopamine receptor protein Important information can be obtained from the structural analysis of purified receptors but purification of the D2 dopamine receptor has proved relatively difficult. The receptor can be solubilized with a good yield from brain and anterior pituitary using detergents 2, but the soluble receptor becomes unstable during purification and this has hindered attempts at purification. Nevertheless, under suitable conditions the soluble receptor can be successfully purified. The D2 dopamine receptor represents only about 0.002% of the protein in receptor-bearing tissues so that powerful affinity chromatography methods are required for purification of the receptor. Several affinity matrices have been developed based on the drugs haloperidol or spiperone linked to sepharose (reviewed in Refs 2,3), and recently three groups have reported purification of the D2 dopamine receptor to apparent homogeneity from brain or pituitary 3-5 using these affinity matrices. The receptor purified from brain runs as a major species on SDS polyacrylamide gel electrophoresis (SDS PAGE) with a molecular mass of 92-95 kDa 3,4. Binding of [3H]spiperone in the purified preparation has the correct pharmacological profile, and the specific activity is about 25% of the theoretical value for a protein of this molecular size. In our own study on the receptor from bovine brain 3, the purified material (molecular mass, 95 kDa) frequently runs as a doublet and this may reflect heterogeneous glycosylation (see below). The receptor purified from anterior pituitary runs as a major species on SDS PAGE of molecular mass 120 kDa 5. Both species of receptor protein are glycoproteins as shown by their ability to bind to lectins. These differences in molecular mass are of added interest in the light of data

© 199o, ElsevierSciencePublishersLtd,(UK)

0166- 2236/90/$02.00

Philip 6. Strange is at the Biological Laboratory, The University, Canterbury CT2 7N], UK.

373

obtained from photoafflnity labelling studies on D2 dopamine receptors, which have shown labelling of 118-150 kDa species in brain and pituitary of some animals in addition to the 95 kDa species most commonly labelled~9. The relevance of these different molecular sizes for the D2 dopamine receptor purified from brain or anterior pituitary is at present unclear. Possible explanations include differential proteolysis or hydrolysis of oligosaccharides; one intriguing possibility is that they represent distinct isoforms of the receptor. Purification of the D2 dopamine receptor from four different bovine brain regions and pituitary using a haloperidol affinity column shows in all cases a major species of 95 kDa. This indicates no significant variation in the receptor species purified by this method, whereas purification of receptors from anterior pituitary using a spiperone affinity column purifies an additional 145 kDa species (Chazot, P. L. and Strange, P. G., unpublished observations). In the next section evidence is presented that the different values for molecular mass observed in some experiments are a result of differential glycosylation of a common core protein. To what extent the variations in protein structure uncovered by gene cloning and outlined below contribute to differences in molecular mass will need to be carefully investigated. The availability of purified receptor from several tissues enables a full structural analysis of the receptor protein to be performed using a variety of protein chemical techniques.

Glycoprotein nature of the D2 dopamine receptor In common with other neurotransmitter receptors, the D2 dopamine receptor is a glycoprotein as shown by its ability to interact with sepharose-bound lectins such as wheat germ agglutinin2,lo. The oligosaccharide chains do not, however, appear to affect greatly the interaction of ligands with the receptor. The structure of the oligosaccharide chains has been investigated by differential adsorption to lectins and by digestion with glycosidase enzymes7-n (Williamson, R. A. and Strange, P. G., unpublished observations). These studies have shown that the D2 dopamine receptor contains a protein core with an estimated molecular mass of 40-44 kDa for deglycosylated photoaffinity labelled receptor and 47-50 kDa for deglycosylated purified receptor. These values for molecular mass are in good agreement with the values derived from the amino acid sequence (see below). Oligosaccharide chains (N-linked) are then attached to this core protein; the glycosylated species show a wide variation in molecular mass values (94-150 kDa) depending on the tissue and the analytical technique used. In the receptor purified from brain, the oligosaccharides are all of the complex type and there is evidence for terminal sialic acids and additional Nacetylglucosamine sequences. In that from the anterior pituitary the oligosaccharides are of the complex and high mannose type. In receptor from both tissues there is relatively heavy sialylation which leads to artefactual behaviour of the receptors on SDS polyacrylamide gel electrophoresis; differential removal of sialic acids may also contribute to some of the heterogeneity in molecular mass values. 374

Chemical modification of the D2 dopamine receptor A fruitful approach to the identification of important amino acid residues at the active sites of enzymes has been chemical modification with group-specific reagents. This approach has been applied to the D2 dopamine receptor 12. A range of group-specific reagents was tested for their effects on [3H]spiperone binding to D2 dopamine receptors, and significant effects were seen with the histidine-specific reagent diethylpyrocarbonate and with the carboxyl-specific reagent dicyclohexylcarbodiimide (DCCD). It has been shown clearly that DCCD is modifying a carboxyl group at the active site of the receptor as the effect can be blocked by both agonists and antagonists; this was not possible for diethyl pyrocarbonate. It is tempting to speculate that the carboxyl group corresponds to the aspartic acid residue implicated in the binding of agonists and antagonists to other monoamine receptors 13 (see below). Further evidence for the importance of a carboxyl group in ligand binding is afforded by the effects of pH on [3H]spiperone binding. Binding of [3H]spiperone is strongly but reversibly inhibited by reducing the pH from 7.4 to 4. The effect corresponds to the titration of a single group of pKa 5.2, which would be consistent with a carboxyl group. More detailed analysis of the effect of pH on drug binding to the D2 dopamine receptor has provided evidence for a second ionizable group that specifically affects the binding of substituted benzamide drugs, e.g. clebopride. The pKa of this group is not known with precision but lies in the range of 6-7. This could be a second carboxyl group or a histidine residue. Similar findings with several ionizing groups affecting antagonist binding have been reported for cardiac muscarinic acetylcholine receptors TM.

Cloning of the D2 dopamine receptor Research on the D2 dopamine receptor has been revolutionized by the report of Bunzow et al. on the isolation of a cDNA corresponding to the 02 dopamine receptor 15. Several of the groups who pursued receptor isolation (see above) were aiming to obtain enough pure receptor so that limited amino acid sequence could be obtained. Oligonucleotide probes based on the amino acid sequence would then have been used to screen cDNA libraries to obtain the cDNA that corresponded to the receptor. Bunzow e t a / . 15, however, took an alternative approach exploiting the significant homologies within the G protein-linked receptor family. Low stringency screening of a rat genomic library using a hamster 132adrenergic receptor probe gave several positive clones. One of these (RGB-2) was used to screen a rat brain cDNA library. This gave an RGB-2 cDNA clone of about 2.5 kb. Northern blot analysis showed that the RGB-2 mRNA was expressed at different levels in different rat brain regions and that the distribution corresponded to that of the D2 dopamine receptor. The cDNA was expressed by transfection into Ltk- mouse fibroblasts and the stable transfectants expressed D2 dopamine receptors (as assessed by [3H]spiperone binding with an appropriate pharmacological profile). The longest open reading flame of the RGB-2 cDNA encodes a 415 amino acid protein (47.064 TINS, Vol. 13, No. 9, 1990

kDa). This is quite close to the size of the deglycosylated form of the D2 dopamine receptor noted above. When the amino acid sequence is analysed for hydropathicity seven stretches of hydrophobic amino acids are seen. Thus the D2 dopamine receptor is predicted to contain seven membrane-spanning o~-helices in common with the other members of the G protein-linked receptor family16,17 whose amino acid sequences are known. This homology is apparent at the level of individual amino acids and the greatest homology is seen in the putative transmembrane domains where up to 40% homology is seen with other G protein-linked receptors, e.g. 0~2-, [~2adrenergic and 5-HT1A-serotonergic. Other homologous features include consensus glycosylation sequences in the predicted extracellular amino terminus, a potential protein kinase A phosphorylation site in the third intracellular loop and a potential site for palmitoylation in the carboxy-terminal tail. A speculative model is shown in Fig. 1, which shows the predicted transmembrane topology. The predicted C-terminal tail of the receptor is notably short, whereas the third intracellular loop is rather long compared with the [52adrenergic receptor and it has been proposed ~7 that this may be typical of receptors that are coupled not to Gs and adenylate cyclase stimulation, but to other G proteins and effectors. The availability of cloned nucleic acid and hence amino acid sequence has led to further significant findings, which we shall consider under three headings, receptor heterogeneity, receptor distribution and receptor-ligand interaction.

Receptor h e t e r o g e n e i t y The existence of multiple post-receptor mechanisms (see above) might suggest that subtypes of the D2 dopamine receptor exist. If these were pharmacologically distinct and tissue-specific this would be of considerable interest for drug design. There has been a flurry of activity in molecular biological studies of this topic and this has led to reports of the cloning of the human homologue of the clone described above together with the discovery in rat, bovine and human tissues of a second mRNA encoding a D2 dopamine receptor 18-26. This second mRNA encodes a slightly longer/larger protein. The amino acid sequences of the corresponding rat, bovine and human receptors are highly homologous (greater than 95% homology at the amino acid level). Within each species the longer and shorter isoforms are identical except for a 29 amino acid insertion in the region corresponding to the third intracellular loop. This insertion is identical in human, bovine and rat receptors except for one amino acid change at the C terminus. The mRNA corresponding to the longer isoform is present at a higher level than that corresponding to the shorter isoform in brain and pituitary. The two receptor forms have been expressed in cultured cells and both forms inhibit adenylate cyclase18. The pharmacological profiles of the two receptor forms in ligand-binding assays with antagonists seem very similar2°,23, although, as yet, not many compounds have been tested; this similarity is not surprising as the 29 amino acid insertion occurs in a region of the receptor not normally associated with the ligand-binding site. However, it would be premaTINS, Vol. 13, No. 9, 1990

ture to draw definitive conclusions on this point and small differences in the binding of some antagonists may eventually be detected. It is interesting that there is high homology between D2 dopamine receptors from different species as some disparity in antagonist binding affinities has been reported in comparisons of bovine and rat receptors 27. This is particularly evident for the substituted benzamide drugs and ought ultimately to be explicable in terms of the amino acid differences in the two species. According to current models of G protein-linked receptors, the part of the receptor containing the 29 amino acid insertion, the third predicted intracellular loop, is associated with the interaction with the G protein. Therefore, it is possible that the absence or presence of the 29 amino acid insertion may alter the structure of this part of the receptor so that the longer and shorter isoforms of the receptor interact with G proteins differently and perhaps interact selectively with distinct G proteins. Consistent with this are minor differences in coupling to adenylate cyclasexs, in agonist binding and in sensitivity to guanine nucleotides that have been reported for the two isoforms expressed in cultured cells2°. These differences are reminiscent of similar differences reported in striatal and limbic regions of bovine brain 2s so perhaps there are differential distributions of the two isoforms in different brain regions (see below). However, the third intracellular loop of G protein-coupled receptors is thought to be important in receptor phosphorylation linked to desensitization17, so the absence or presence of the 29 amino acid insertion may also affect the regulation of receptor number in the membrane. For many of the G protein-linked receptors the entire polypeptide is encoded by one large exon but in the original report of the cloning of the rat D 2 dopamine receptor some evidence for introns in the receptor gene was provided ~5. For the human gene a complex arrangement of several introns and exons is seen TM with seven exons in the coding region23,29. Interestingly, the 29 amino acid insertion that differentiates the two isoforms of the receptor is encoded by a single exon so that alternative splicing is likely to underlie the existence of the two forms of receptor. This is the first example in the G protein-linked receptor family where alternative splicing is used to generate diversity. For other neurotransmitters, gene duplication has been observed as the source of diversity. In the case of the D2 dopamine receptor there seems to be only one gene 23 so that alternative splicing must be used to generate diversity. If this diversity relates only to variants in the third intracellular loop as described here then the prospects for pharmacologically highly distinct receptor subtypes are bleak. If other variants, with alternative splicing of other regions, are also seen then perhaps pharmacologically distinct subtypes will be observed. In this context an expression cloning approach has identified a rat D2 dopamine receptor gene that seems to be different from the original gene coding for the shorter isoform 3°. The ligand-binding properties of the expressed protein are different from those of either the longer or shorter isoform of the receptor. Further work will be required to establish whether this represents a novel additional receptor subtype and if so, what its origin is. 375

Receptor distribution

(PCR) 2°,29 and this suggests some tissue specificity in distribution. In pituitary and basal ganglia, mRNA for the longer isoform predominates, but in other brain regions it is mRNA for the shorter isoform that is more represented or predominates. It will be of great interest to determine whether these relative distributions are reflected at the protein level and whether the relative distribution alters in diseases like schizophrenia. If we consider in more detail the receptors being labelled in these studies then, for example, for the mesostriatal dopamine pathway, labelling in the striatum must correspond to labelling of postsynaptic receptors, whereas that in the substantia nigra must correspond to labelling of autoreceptors. There are no data at present to suggest preferential localization of the two isoforms in the different regions; this is consistent with postsynaptic receptors and autoreceptors having similar or identical antagonist pharmacological profiles. Differences in agonist sensitivity at postsynaptic receptors and autoreceptors36 would then reflect different numbers of spare receptors rather than different receptor species. In situ hybridization also enables receptor mRNA to be localized at the cellular level. The data are not extensive as yet but show labelling in the striatum of the predominant medium-sized spiny cell, which is expected to be the site of the postsynaptic D2 receptor 37, as well as some labelling of other cells Extracellular which may be cholinergic interneurones. Determining the precise distribution of receptors on different cell types will help clarify the functions of dopamine in the striatum.

The distribution of D2 dopamine receptors between tissues and at the cellular level is of some interest as it may shed further light on the functions of dopamine. This has been examined using receptor-ligand autoradiography but the availability of cloned nucleic acid sequence allows in situ hybridization to be carried out to localize receptor mRNA. Several studies on this have recently been published using oligonucleotide probes that would detect mRNA corresponding to both isoforms of the receptoral-35. Generally, the results are in agreement with data from receptor autoradiography: the highest levels of receptor mRNA are found in the substantia nigra pars compacta and ventral tegmental area, where the cell bodies of the mesostriatal and mesocortical dopamine system are found, and in the striatum and certain limbic regions (olfactory tubercle, nucleus accumbens), which are the areas of termination of these neurones. Some signal was detected in the regions of the cerebral cortex receiving a dopamine innervation and mRNA was also detected in the anterior and intermediate lobes of the pituitary. The relative abundances of mRNA for the two isoforms have been examined using the polymerase chain reaction

A

oot,,)1

B

~so) O=G :

1

2

Intracellular Fig. 1. Models for the structure of the D2 dopamine receptor. (A) Model of the disposition of the D2 dopamine receptor polypeptide as predicted by hydropathicity analysis. The numbered rectangles are the predicted transmembrane ol-helical regions linked by loops of varying length outside the membrane. Of note are the long loop (135 amino acids in the shorter isoform, 163 amino acids in the longer isoform) linking the fifth and sixth transmembrane domains, the extracellular amino-terminal section with potential glycosylation (Y) sites and the short (14 amino acid) carboxy-terminal tail. The carboxy-terminal cysteine residue may be palmitoylated 43 in which case the carboxyterminal tail could associate with the membrane forming a fourth intracellular loop. The dashed line in the third predicted intracellular loop represents the 29 amino acid insertion that differentiates the longer and shorter isoforms of the receptor. Transmembrane domains 2, 4, 5, 6 and 7 contain proline residues so the rectangular representation is misleading and there is likely to be significant deviation from ~-helical structure. Cysteine residues in the loops linking transmembrane domains 2 and 3, and 4 and 5 may form a disulphide bond important for the tertiary structure of the receptor. The figure shows the approximate positions of the conserved aspartic acid (D) residues that may be important for ligand binding. Their role is discussed in detail in the text. (B) By analogy with other G protein-linked proteins, the transmembrane domains are likely to be bundled and this is a view from the intracellular face of the membrane of how this might occur based on the model of Findlay and Pappin 38 for rhodopsin. The transmembrane domains are shown in cross-section leaving a cavity into which ligands may bind by interaction with specific amino acid side-chains, and dopamine is shown speculatively interacting with the aspartic acid (114) in the third domain (see text). The diagram is misleading in one respect in that it does not show the depth of the cavity across the membrane; it is likely to be at least as deep as it is wide at its largest width. There is, therefore, substantial scope for movement of ligands in the transmembrane dimension. 376

~NS, Vol.13, No.~ 1990

Receptor-ligand interaction Exactly how the De dopamine receptor binds ligands and for agonists how this leads to transmembrane signalling is a critical question. For the D2 dopamine receptor the ligand-binding site has not yet been delineated but, by analogy with the models proposed for rhodopsin and for the [3-adrenergic receptor 13,38, it is likely to be formed by the bundling of the putative transmembrane regions; Fig. 1 attempts to show how this might occur. The cavity formed by the helices would then contain the binding site with certain strategic amino acid side-chains forming contacts with the ligands. The binding site of the receptor has to accept both antagonists and agonists and the latter must elicit a productive conformafional change in the receptor leading to receptor activation. The basis of the differences between agonist and antagonist binding are not clear but must involve differential interactions with amino acid residues at the active site. Owing to the cationic nature of both agonist and antagonist ligands a primary interaction is likely to be electrostatic with a negatively charged residue at the active site. Other interactions, for example hydrophobic interactions and those via hydrogen bonds, must contribute differentially to agonist and antagonist binding. For catecholamine agonists a hydrogen-bond interaction with the two catechol hydroxyl groups is expected la. Antagonists tend to be larger and more hydrophobic and so hydrophobic interactions with the receptor will be important. The range of chemical structures of D2 dopamine antagonists is impressive, but eventually a detailed description of the specific interactions of the different chemical structures with the receptor will be possible. Some clues to the amino acid residues involved in ligand-receptor interaction may be obtained by looking for amino acids in the predicted transmembrane regions that would be negatively charged at neutral pH. Those found in the D2 dopamine receptor and conserved in other receptors that bind cationic amines would be of particular interest. This kind of analysis highlights Asp 80 in predicted membrane-spanning region 2 and Asp 114 and Asp 131 in or near membrane-spanning region 3, analogous aspartic acid residues being found in other receptors that bind cationic amines. Asp 108, also near transmembrane region 3, corresponds to aspartic or glutamic acid in most (but not all) of the other receptors that bind cationic amines and so may be of interest. In Fig. 1 I have indicated speculatively the rough positions of these residues within the membrane, and it can be seen that they provide a series of negative charges spanning the membrane. The roles of these residues in the function of the D2 dopamine receptor will remain unknown until mutagenesis studies have been performed, but we can make analogies with the 13-adrenergic and muscarinic receptors where such studies have been carried out 13'39'40. For both [3-adrenergic and muscarinic receptors antagonist binding is dominated by a single aspartic acid residue corresponding to Asp 114 of the D2 dopamine receptor. Asp 114 would then act as the counterion for antagonists such as spiperone in binding to the D2 dopamine receptor, and modification of Asp 114 may be important for the effects of DCCD TINS, Vol. 13, No. 9, 1990

(see above). In [3-adrenergic receptors the same aspartic acid residue is important for agonist binding. Mutagenesis of the residues corresponding to Asp 80 in the D2 dopamine receptor does not affect antagonist binding but does affect agonist binding, with opposite effects in the [3-adrenergic and muscarinic receptors. Agonists and antagonists therefore bind primarily to the residue corresponding to Asp 114 but for agonists the residue corresponding to Asp 80 also affects agonist binding 13. Asp 114 (or equivalent) is present only in receptors that bind cationic amines, reinforcing the importance of this residue. Residues corresponding to Asp 80 are present in almost all the G protein-coupled proteins suggesting that this residue may have a role in the transduction mechanism or in maintaining receptor structure rather than a primary role in binding cationic amines. The residue corresponding to Asp 131 is also highly conserved and found in pepfide receptors as well as those binding cationic amines. It is also adjacent to an arginine and so likely to be ion-paired. This again argues against a primary role in ligand binding. Mutagenesis of this residue in the ~-adrenergic and muscarinic receptors does not affect antagonist binding but produces increases in agonist affinities. These findings and the proximity of the residue to the intracellular face of the receptor argue for a role in transduction. Mutagenesis of the acidic residue corresponding to Asp 108 affects both agonist and antagonist binding to the muscarinic receptor 4° but has no effect for the 13adrenergic receptor 41 so that this residue may participate in ligand binding, perhaps by facilitating the access of the cationic ligand to the receptor site. The charged residues at the outer and inner faces of the membrane could also play roles in the biosynthesis of these proteins acting as part of the internal signal or stop-transfer sequences of the proteins 42. In summary, then, ligand binding to the receptors that bind cationic amines is likely to be dominated by an electrostatic interaction with the residue corresponding to Asp 114 of the D2 dopamine receptor. Residues corresponding to Asp 80 and Asp 108 also participate to varying degrees in binding of agonists and antagonists. Therefore agonist and antagonist binding configurations are not identical but are likely to overlap. The different pH dependencies of certain antagonists for binding to muscarinic 14 and D2 dopamine receptors 12 also argue for specific modes of binding of different antagonists. If certain antagonists bind to the receptors in slightly different ways, then the ionizing groups at the receptor site will have differential effects on their binding.

Selected references 1 Vallar, L. and Meldolesi, J. (1989) Trends Pharmaco/. Sci. 10, 74-77 2 Strange, P. G. (1987) in Dopamine Receptors (Creese, I. and Fraser, C. M., eds), pp. 29-44, Alan R. Liss 3 Williamson, R. A., Worrall, S., Chazot, P. L. and Strange, P. G. (1988) EMBO J. 7, 4129-4133 4 Elazar, Z., Kanety, H., David, C. and Fuchs, S. (1988) Biochem. Biophys. Res. Commun. 156, 602-609 5 Senogles, S. E., Amlaiky, N., Falardeau, P. and Caron, M. G. (1988) J. Biol. Chem. 263, 18996-19002 6 Amlaiky, N. and Caron, M. G. (1986) J. Neurochem. 47, 196-204 7 Grigoriadis, D. E., Niznik, H. B., Jarvie, K. R. and Seeman, P. (1986) FEBSLeft. 227, 220-224 377

Acknowledgements I thank my colleagues in Canterbury for many helpful discussions,John Findlayand Nigel Birdsall for helpful advice and Sarah 5il/ing and Sue Davies for preparing the manuscript.

8 Jarvie, K. R., Niznik, H. B. and Seeman, P. (1988) Mol. Pharmacol. 34, 91-97 9 Jarvie, K. R. and Niznik, H. B. (1989)J. Biochem. 106, 17-22 10 Abbott, W. M. and Strange, P. G. (1985) Biosci. Rep. 5, 303-308 11 Leonard, M. N., Williamson, R. A. and Strange, P. G. (1988) Biochem. J. 255, 877-883 12 Williamson, R. A. and Strange, P. G. J. Neurochem. (in press) 13 Strader, C. D., Sigal, I. S. and Dixon, R. A. F. (1989) FASEBJ. 3, 1825-1832 14 Birdsall, N. J. M., Chan, S., Eveleigh, P., Hulme, E. C. and Miller, K. (1989) Trends Pharmacol. Sci. 10 (Suppl.), 31-34 15 Bunzow, J. R. etal. (1988) Nature 336, 783-787 16 Strange, P. G. (1988) Biochem. J. 249, 309-318 17 Lefkowitz, R. J. and Caron, M. G. (1988)J. Biol. Chem. 263, 4993-4996 18 Dal Toso, R. etal. (1989) EMBOJ. 13, 4025-4034 19 Selbie, L. A., Hayes, G. and Shine, J. (1989) DNA 8, 683-689 20 Giros, B. et al. (1989) Nature 342,923-926 21 Monsma, F. J., McVittie, L. D., Gerfen, C., Mahan, L. C. and Sibley, D. R. (1989) Nature 342,926-929 22 Eidne, K., Taylor, P. L., Zabavnik, J., Saunders, P. T. K. and Inglis, J. D. (1989) Nature 342, 865 23 Grandy, D. K. et a/. (1989) Proc. Natl Acad. Sci. USA 86, 9762-9766 24 Miller, J. C., Wang, Y. and Filer, D. (1990) Biochem. Biophys. Res. Commun. 166, 109-112 25 Chio, C. L., Hess, G. F., Graham, R. S. and Huff, R. M. (1990) Nature 343, 266-269 26 Stormann, T. M., Gdula, D. C., Weiner, D. M. and Brann, M. R. (1990) 4,1ol. PharmacoL 37, 1-6 27 Leonard, M. N., Halliday, C. A., Marriott, A. S. and Strange, P. G. (1988) Biochem. Pharmacol. 37, 4335-4339

28 Leonard, M. N., Macey, C. A. and Strange, P. G. (1987) Biochem. J. 248, 595-602 29 O'Malley, K. L., Mack, K. J., Gandelman, K. and Todd, R. (1990) Biochemistry 29, 1367-1371 30 Todd, R. D., Khurana, T. S., Sajovic, P., Stone, K. R. and O'Malley, K. L. (1989) Proc. Nat/ Acad. Sci. USA 86, 10134-10138 31 Najlerahim, A., Barton, A. J. L., Harrison, P. J., Heffernan, J. and Pearson, R. C. A. (1989) FEBS Left:. 255, 335-339 32 Meador-Woodruff, J. H. et al. (1989) Proc. Natl Acad. Sci. USA 86, 7625-7628 33 Mengod, G., Martinez-Mir, M. I., Vilaro, M. T. and Palacios, J. M. (1989) Proc. NatlAcad. Sci. USA 86, 8560-8564 34 Weiner, D. M. and Brann, M. R. (1989) FEBS Lett. 253, 207-213 35 Le Moine, C. et al. (1990) Proc. Nat/Acad. Sci. USA 87, 230-234 36 Cooper, J. R., Bloom, F. E. and Roth, R. H. (1986) Biochemical Basis of Neuropharmacology Oxford University Press 37 Strange, P. G. (1990) Trends Neurosci. 13, 93-94 38 Findlay, J. B. C. and Pappin, D. J. (1986) Biochem. J. 238, 625-642 39 Venter, J. C., Fraser, C. M., Kerlavage, A. R. and Buck, M. A. (1989) Biochem. Pharmacol. 38, 1197-1208 40 Fraser, C. M., Wang, C., Robinson, D. A., Gocayne, J. D. and Venter, J. C. (1989) Mol. PharmacoL 36, 840-847 41 Strader, C. D. et al. (1987) Proc. Natl Acad. Sci. USA 84, 4384-4388 42 Audigier, Y., Friedlander, M. and Blobel, G. (1987) Proc. Natl Acad. Sci. USA 84, 5783-5787 43 O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J. and Bouvier, M. (1989) J. BioL Chem. 264, 7564-7569

Calciumand the mechanismof light adaptationin vertebrate photoreceptors Gordon

6ordon L. Fainis at the Department of Ophthalmology, Jules Stein Eye Institute, UCLA Schoolof Medicine, Los

Angeles,CA90024, USA and Hugh R. Matthews is at the Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

378

L. Fain a n d H u g h R. M a t t h e w s

Vertebrate photoreceptors transduce the absorption of light into a hyperpolarizing change m membrane potential. The mechanism of transduction is becoming fairly well understood and has been shown to occur via a G protein-coupled decrease in cyclic GMP. Attention is now turning to the way the enzymatic machinery in the outer segment of the photoreceptor cell is modulated during light adaptation. Recent studies show that light adaptation cannot occur if changes in the concentration of cytoplasmic free calcium in the outer segment are prevented, suggesting that calcium functions as a second messenger in sensitivity regulation. A significant achievement of neuroscience during the past decade has been the elucidation of the mechanism of excitation in vertebrate photoreceptors. We now know, or believe we know each of the steps that occurs, from the absorption of a photon to the change in potential across the photoreceptor plasma membrane. The internal messenger that regulates the opening and closing of the light-dependent conductance has now clearly been shown to be cGMP (see Ref. 1). The proteins involved in visual excitation have been characterized in considerable detail; most have been isolated and purified, and many have been cloned and sequenced (see Ref. 2). The techniques of molecular biology, in particular site-directed muta-

genesis, are currently being used to elucidate the mechanism of photon capture and the nature of the protein-protein interactions responsible for signal transduction (see, for example, Ref. 3). As understanding of the mechanism of excitation increases, attention is turning to the ways in which transduction is modulated. Light exposure, in addition to producing excitation, also initiates a sequence of events that alters the sensitivity and kinetics of transduction ~6. These events, collectively referred to as light adaptation, enable the photoreceptor to adjust its sensitivity according to the intensity of the ambient illumination. They seem to occur as the result of regulatory changes in the mechanisms that control cyclic nucleotide levels in the photoreceptor outer segment. Although we do not understand in detail how adaptation occurs, recent experiments indicate that calcium (Ca 2+) plays an important role and probably serves as an internal messenger for sensitivity modulation 7-n. The excitatory cascade In order to describe possible mechanisms that might be responsible for adaptation, it is first necessary to describe briefly the enzymatic cascade that produces the electrical response (see Fig. 1A and Refs 2, 12-15). Excitation begins with the absorption

© 1990,ElsevierSciencePublishersLtd,(UK) 0166- 2236/90/$02.00

TINS, VoL 13, NO. 9, 1990

Aspects of the structure of the D2 dopamine receptor.

Significant new information on the D2 dopamine receptor has recently become available from a combination of protein chemical and molecular genetic ana...
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