TiPS - November 2992 [Vol. 13J
399
Dopamine:FromCinderella to HolyGrail In the opening plenary lecture of a recent meeting* Julius Axelrod (NIMH, Bethesda) recalled a time when dopamine was considered a mere precursor molecule, the Cinderella of the catecholamine family. Nowadays dopamine is firmly established as a neurotransmitter molecule in its own right. However, the discovery in the past few years of new dopamine receptor subtypes* has added complexity to what had seemed a comparatively simple neurotransmitter system. Of special interest to many pharmacologists are the DP-like receptors, Da and Da, Antagonism of D2 receptors is the mode of action for many present-day antipsychotic drugs. Pierre Sokoloff (INSERM, Paris) and his group have extensively studied the properties of the D3 receptor recombinantly expressed in Chinese hamster ovary (CHO) cells. However, they have been unable to identify the signal transduction mechanisms associated with this receptor. Unlike many seven-transmembrane-domain receptors, the Da receptor, in both CHO cells and cerebellum, exhibits only weak shifts of agonist binding in the presence of guanine nucleotide. Although this could be interpreted as indicating a lack of G protein-coupling, Sandra Castro (University of Kent, Canterbury) has shown that D3 receptors transfected into a similar CHO cell line show high- and low-affinity binding sites for agonists such as dopamine (Fig. 1) and apomorphine. As expected, there is a complete shift to lowaffinity binding in the presence of GTE and work is underway to find the biochemical correlate of the high-affinity site. More direct evidence for a functionally active recombinant D3 receptor came from the work of Richard Todd (Washington University, St Louis). His group used a CNS-derived cell line, MN9D “7th International posium, Amsterdam,
Catecholamine ]une 22-26 2992.
Sym-
(Ref. 2), to stably express the receptor. When Ds-transfected MN9D cells are incubated with the dopamine receptor agonist quinpirole, distinct morphological changes occur. The number of branch-points per cell increases by 300% and there is also a small increase in the length of the processes. Conversely, MN9D cells transfected with the Da receptor show only a small rise in the number of branch-points but a 50% elongation of the processes. No change in either parameter was seen with Dz transfected cells. As yet, the signal transduction
pathways involved in this transformation have not been identified although Da receptors in this cellular environment do not appear to couple to either CAME formation or phosphoinositide hydrolysis. In the fhury of act++ that followed the initial cloning of the D2 receptor, a second isoform was identified in human3, bovine4 and rats brain. Identical except for an insert of 29 amino acid:.. these receptor protein isofonns arise from differential splicing of the Da mRNA, not as the products of separate genes. In a comprehensive study involving 25 antagonists from 11 different structural classes, Josie Leysen (Janssen Research Foundation, Beerse) investigated the pharmacology of the short (Dx) and 1oi.g (DrL) forms of the 4 receptor transfected into HER293 cells. No dif-
110 1
I
0
80 -
e 8
80-
30 20 10 I
-9
I
-8
I
I
-7
-8
Log [dopamine]
(M)
Fig. 1. Dopamine compelition for ~H]spiperone binding to mtwbranes of ,%ombinant CHO cells expressing the rat D3 dopamine recaptor gene. In the absenca of GTP (O), the competition curve fits best to a iwo site mode/ wheraas in the presence of GTP (llW,w, +) a one-site model best fits the data (Castm, S. W and Strange, P. G.. unpublished). .m.. @1992, ElsevierSciencePublishers
Ltd (UK)
Tips - November 1992 fV01.131 ferences were observed in binding affinities. In direct contrast to this, Philip Strange (University of Kent, Canterbury) reported that the same receptors, expressed in Ltk- fibroblast cells, have significantly different pharmacological profiles. while classical dopamine such as receptor antagonists spiperone, butaclamol and haloperidol gave Ki ratios (D&&) of -1, substituted benzamide compounds gave values ranging from 1.5 for remoxipride to a value of -5 for DO7lO. The signal transduction pathw;tys linked to the two DZreceptor &oforms were analysed in GH& and Ltk- cells by Lucia Vallar (Scientific Institute San Raffaele, Milan). Both isoforms were found to be equally effective at producing an inhibition of adenylyl cyclase, stimulation of phosphoinositide hydrolysis (Ltk-) or a decrease in intracellular Ca” levels (GH&). The G proteins associated with each isoform were identified using a labelling technique in which cholera toxin ADP-ribosylates receptor-activated G proteins. For both DE and DZL in G&C1 cells, agonist stimulation resulted in the ADP-ribosylation of the alpha subunits of Gi2, Gi3 and G,,. The same profile of G protein usage is seen in the Ltkcells but with different labelling intensities, reflecting the divergent signalling pathways. These results are somewhat unexpected as the two forms of the receptor vary only in the third cytoplasmic loop. This is a region implicated in G protein-coupling rather than ligand binding. However, it is possible that the insertion of 29 amino acids in the intracellular portion of the receptor results in conformational changes (Strange) that influence the binding characteristics of the receptor. Although mRNA splice variants have been found for DZ and D3 receptor@*’ none have yet b?en identified for D4, the other member of the D2 receptor subfamily. However, Hubert Van To1 (Clarke Institute of Psychiatry, Toronto) reported the presence of a polymorphic sequence of 48 base pairs that appears 2, 4 or 7 times in the putative third intracellular loop of the D4 receptor. These repeated sequences were originally thought to be an intron, but analysis of human genomic
DNA has revealed them to be a true polymorphism. When expressed in COS-7 cells these Da receptor variants display a marked change in their pharmacology, particularly as regards the NaCl sensitivity of clozapine binding. Clozapine is an atypical neuroleptic drug that is the most common treatment currently used for schizophrenia. Van To1 and colleagues have speculated that this receptor polymorphism may underlie a susceptibility to psychiatric disorders or determine whether a patient will respond to clozapine therapy. A number of studies have appeared recently in which the levels of dopamine receptor mRNA following antipsychotic treatment have been measured. Ian Geese (State University of New Jersey, Newark) presented data from rat brain that demonstrated that mRNA levels cannot always be directly correlated with the level of the corresponding receptor protein. Upregulation of dopamine receptors by denervation, dopamine depletion or chronic receptor blockade could only be matched to equivalent increases in receptor mRNA levels in the case of denervation. Blockade of D2 receptors by treatment with haloperidol for up to 21 days caused a significant increase in receptor number that was not related to changes in receptor transcription. Further analysis revealed that the rise in receptor level resulted from a reduction in the rate of D2 receptor degradation. It would appear from this data that quantitation of receptor changes following neuroleptic treatment will require direct measurement of the receptor proteins using subtype-specific ligands. The need for selective ligands was also apparent in the work of Nancy Zahniser (University of Colorado, Denver) on the dopamine autoreceptor in rat striatum. The D3 receptor has high affinity for the putative autoreceptor antagonists UH232 and AJ76 (Ref. 8) and this has led to the suggestion that it may be an autoreceptor. Using striatal slices in a superfusion system, the ability of dopamine agonists to inhibit electrically-evoked dopamine release was measured. This was compared to the agonists’ affinities
in a binding assay using the same tissue, where the dominant receptor activity detected is DP. The rank order of potency for three agonists was the same in both assays, indicating that the autoreceptor in this tissue resembles D2 rather than Ds. However, in the absence of selective agonists it is difficult to pursue this observation further.
0
q
0
There is a pressing need, in both biochemical and pharmacological studies, for compounds able to differentiate between members of the Dz-like receptor subfamily, but unfortunately the outlook is not encouraging. The ability of known dopamine receptor-active compounds to discriminate between D2 and D3 receptors is poor (Leysen) and the majority have yet to be tested on D4 receptors. Development of a chemical structure selective for one of these closely related receptor subtypes may prove to be the next Holy Grail for medicinal chemists. ANN MILLS
Institute for Molecular Biology, 24 chemin des Aulx, 2228 Plan-les-Ouatesl Genevn, Switzerfnnd. Glaxo
References 1 Sibley, D. R. and Monsma, F. J. (1992) Trends Pharmacol. Sci. 13, 61-69 2 Choi, H. K. et al. (1991) Bruin Res. 552, 67-76 3 Grandy, D. K. et al. (1989) Proc. Nutl Acad. Sci. USA 86.9762-9766 4 Chio, C. L., Hess, G. F., Graham, R. S. and Huff, R. M. (1990) Nature 343, 266-269 5 Rao, D. D., McKelvy, J., Kebabian, J. and MacKenzie, R. G. (1990) FEBS Lett. 263(l), 18-22 6 Giros, B., Martres, M-P., Pilon, C., Sokoloff, P. and Schwartz, J-C. (1991) Biochem. Biophvs. Res. Commun. 176(3),. 1584-1592 ’ ” 7 Snyder, L. A., Roberts, J. L. and Sealfon, S. C. (1991) Biochek. Biophys. Res. Commun. 180,1031-1035 8 Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J. C. (1990) Nature 347,146-150
AJ76: cis-(+)-(ls,2R)-5-methoxy-l-methyl2-(n-propylamino) tetralin D0710: N-((l-ethyl-2-pyrrolidinyl)methyl)-2-methoxy-5-((methylamino)sulfonyl)benzamide UH232: cis-(+)-(ls,2R)-5-methoxy-lmethyl-2-(di-n-pmpylamino) tetralin
399
Dopamine:FromCinderella to HolyGrail In the opening plenary lecture of a recent meeting* Julius Axelrod (NIMH, Bethesda) recalled a time when dopamine was considered a mere precursor molecule, the Cinderella of the catecholamine family. Nowadays dopamine is firmly established as a neurotransmitter molecule in its own right. However, the discovery in the past few years of new dopamine receptor subtypes* has added complexity to what had seemed a comparatively simple neurotransmitter system. Of special interest to many pharmacologists are the DP-like receptors, Da and Da, Antagonism of D2 receptors is the mode of action for many present-day antipsychotic drugs. Pierre Sokoloff (INSERM, Paris) and his group have extensively studied the properties of the D3 receptor recombinantly expressed in Chinese hamster ovary (CHO) cells. However, they have been unable to identify the signal transduction mechanisms associated with this receptor. Unlike many seven-transmembrane-domain receptors, the Da receptor, in both CHO cells and cerebellum, exhibits only weak shifts of agonist binding in the presence of guanine nucleotide. Although this could be interpreted as indicating a lack of G protein-coupling, Sandra Castro (University of Kent, Canterbury) has shown that D3 receptors transfected into a similar CHO cell line show high- and low-affinity binding sites for agonists such as dopamine (Fig. 1) and apomorphine. As expected, there is a complete shift to lowaffinity binding in the presence of GTE and work is underway to find the biochemical correlate of the high-affinity site. More direct evidence for a functionally active recombinant D3 receptor came from the work of Richard Todd (Washington University, St Louis). His group used a CNS-derived cell line, MN9D “7th International posium, Amsterdam,
Catecholamine ]une 22-26 2992.
Sym-
(Ref. 2), to stably express the receptor. When Ds-transfected MN9D cells are incubated with the dopamine receptor agonist quinpirole, distinct morphological changes occur. The number of branch-points per cell increases by 300% and there is also a small increase in the length of the processes. Conversely, MN9D cells transfected with the Da receptor show only a small rise in the number of branch-points but a 50% elongation of the processes. No change in either parameter was seen with Dz transfected cells. As yet, the signal transduction
pathways involved in this transformation have not been identified although Da receptors in this cellular environment do not appear to couple to either CAME formation or phosphoinositide hydrolysis. In the fhury of act++ that followed the initial cloning of the D2 receptor, a second isoform was identified in human3, bovine4 and rats brain. Identical except for an insert of 29 amino acid:.. these receptor protein isofonns arise from differential splicing of the Da mRNA, not as the products of separate genes. In a comprehensive study involving 25 antagonists from 11 different structural classes, Josie Leysen (Janssen Research Foundation, Beerse) investigated the pharmacology of the short (Dx) and 1oi.g (DrL) forms of the 4 receptor transfected into HER293 cells. No dif-
110 1
I
0
80 -
e 8
80-
30 20 10 I
-9
I
-8
I
I
-7
-8
Log [dopamine]
(M)
Fig. 1. Dopamine compelition for ~H]spiperone binding to mtwbranes of ,%ombinant CHO cells expressing the rat D3 dopamine recaptor gene. In the absenca of GTP (O), the competition curve fits best to a iwo site mode/ wheraas in the presence of GTP (llW,w, +) a one-site model best fits the data (Castm, S. W and Strange, P. G.. unpublished). .m.. @1992, ElsevierSciencePublishers
Ltd (UK)
Tips - November 1992 fV01.131 ferences were observed in binding affinities. In direct contrast to this, Philip Strange (University of Kent, Canterbury) reported that the same receptors, expressed in Ltk- fibroblast cells, have significantly different pharmacological profiles. while classical dopamine such as receptor antagonists spiperone, butaclamol and haloperidol gave Ki ratios (D&&) of -1, substituted benzamide compounds gave values ranging from 1.5 for remoxipride to a value of -5 for DO7lO. The signal transduction pathw;tys linked to the two DZreceptor &oforms were analysed in GH& and Ltk- cells by Lucia Vallar (Scientific Institute San Raffaele, Milan). Both isoforms were found to be equally effective at producing an inhibition of adenylyl cyclase, stimulation of phosphoinositide hydrolysis (Ltk-) or a decrease in intracellular Ca” levels (GH&). The G proteins associated with each isoform were identified using a labelling technique in which cholera toxin ADP-ribosylates receptor-activated G proteins. For both DE and DZL in G&C1 cells, agonist stimulation resulted in the ADP-ribosylation of the alpha subunits of Gi2, Gi3 and G,,. The same profile of G protein usage is seen in the Ltkcells but with different labelling intensities, reflecting the divergent signalling pathways. These results are somewhat unexpected as the two forms of the receptor vary only in the third cytoplasmic loop. This is a region implicated in G protein-coupling rather than ligand binding. However, it is possible that the insertion of 29 amino acids in the intracellular portion of the receptor results in conformational changes (Strange) that influence the binding characteristics of the receptor. Although mRNA splice variants have been found for DZ and D3 receptor@*’ none have yet b?en identified for D4, the other member of the D2 receptor subfamily. However, Hubert Van To1 (Clarke Institute of Psychiatry, Toronto) reported the presence of a polymorphic sequence of 48 base pairs that appears 2, 4 or 7 times in the putative third intracellular loop of the D4 receptor. These repeated sequences were originally thought to be an intron, but analysis of human genomic
DNA has revealed them to be a true polymorphism. When expressed in COS-7 cells these Da receptor variants display a marked change in their pharmacology, particularly as regards the NaCl sensitivity of clozapine binding. Clozapine is an atypical neuroleptic drug that is the most common treatment currently used for schizophrenia. Van To1 and colleagues have speculated that this receptor polymorphism may underlie a susceptibility to psychiatric disorders or determine whether a patient will respond to clozapine therapy. A number of studies have appeared recently in which the levels of dopamine receptor mRNA following antipsychotic treatment have been measured. Ian Geese (State University of New Jersey, Newark) presented data from rat brain that demonstrated that mRNA levels cannot always be directly correlated with the level of the corresponding receptor protein. Upregulation of dopamine receptors by denervation, dopamine depletion or chronic receptor blockade could only be matched to equivalent increases in receptor mRNA levels in the case of denervation. Blockade of D2 receptors by treatment with haloperidol for up to 21 days caused a significant increase in receptor number that was not related to changes in receptor transcription. Further analysis revealed that the rise in receptor level resulted from a reduction in the rate of D2 receptor degradation. It would appear from this data that quantitation of receptor changes following neuroleptic treatment will require direct measurement of the receptor proteins using subtype-specific ligands. The need for selective ligands was also apparent in the work of Nancy Zahniser (University of Colorado, Denver) on the dopamine autoreceptor in rat striatum. The D3 receptor has high affinity for the putative autoreceptor antagonists UH232 and AJ76 (Ref. 8) and this has led to the suggestion that it may be an autoreceptor. Using striatal slices in a superfusion system, the ability of dopamine agonists to inhibit electrically-evoked dopamine release was measured. This was compared to the agonists’ affinities
in a binding assay using the same tissue, where the dominant receptor activity detected is DP. The rank order of potency for three agonists was the same in both assays, indicating that the autoreceptor in this tissue resembles D2 rather than Ds. However, in the absence of selective agonists it is difficult to pursue this observation further.
0
q
0
There is a pressing need, in both biochemical and pharmacological studies, for compounds able to differentiate between members of the Dz-like receptor subfamily, but unfortunately the outlook is not encouraging. The ability of known dopamine receptor-active compounds to discriminate between D2 and D3 receptors is poor (Leysen) and the majority have yet to be tested on D4 receptors. Development of a chemical structure selective for one of these closely related receptor subtypes may prove to be the next Holy Grail for medicinal chemists. ANN MILLS
Institute for Molecular Biology, 24 chemin des Aulx, 2228 Plan-les-Ouatesl Genevn, Switzerfnnd. Glaxo
References 1 Sibley, D. R. and Monsma, F. J. (1992) Trends Pharmacol. Sci. 13, 61-69 2 Choi, H. K. et al. (1991) Bruin Res. 552, 67-76 3 Grandy, D. K. et al. (1989) Proc. Nutl Acad. Sci. USA 86.9762-9766 4 Chio, C. L., Hess, G. F., Graham, R. S. and Huff, R. M. (1990) Nature 343, 266-269 5 Rao, D. D., McKelvy, J., Kebabian, J. and MacKenzie, R. G. (1990) FEBS Lett. 263(l), 18-22 6 Giros, B., Martres, M-P., Pilon, C., Sokoloff, P. and Schwartz, J-C. (1991) Biochem. Biophvs. Res. Commun. 176(3),. 1584-1592 ’ ” 7 Snyder, L. A., Roberts, J. L. and Sealfon, S. C. (1991) Biochek. Biophys. Res. Commun. 180,1031-1035 8 Sokoloff, P., Giros, B., Martres, M-P., Bouthenet, M-L. and Schwartz, J. C. (1990) Nature 347,146-150
AJ76: cis-(+)-(ls,2R)-5-methoxy-l-methyl2-(n-propylamino) tetralin D0710: N-((l-ethyl-2-pyrrolidinyl)methyl)-2-methoxy-5-((methylamino)sulfonyl)benzamide UH232: cis-(+)-(ls,2R)-5-methoxy-lmethyl-2-(di-n-pmpylamino) tetralin
