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Cloning of a ‘D3’ receptor subtype expands dopamine receptor family
TiPS- january1991 lVo1.121
7
of a ‘Di receptor subtype expands dopamine receptor family
Cloning
Ten years ago, only two different dopamine receptor subtypes were definitively known to exist: the D, receptor, linked to the stimulation of adenylyl cyclase, and the D2 receptor which either inhibited adenylyl cyclase or was unlinked to this enzyme’. DZreceptors have now been suggested to be linked to additional second messenger systems, including inhibition of phosphatidylinositol (PI) tumover, activation of K+ channels and inhibition of Ca2+ channel activityz. The existence of additional DS and Dd receptors was proposed in the early 19&W4, but this was based on differing agonist affinities in ligand-binding assays and subsequently shown to represent different affinity states of the known Dt and D2 receptorss. New information derived from cloning of the D1 and D2 receptors, as well as that of a putative D3 subtype, now indicates that the dopamine receptor family could be even more heterogeneous than previously suspected.
The D1 receptor linked to the activation of adenylyl cyclase has recently been cloned from both rat and human tissues-. This receptor is 446 amino acids in length and has aeven putative membranespanning domains typical of the G protein-linked receptor familylo. When expressed in mammalian cells, the cloned D1 receptor exhibits the expected pharmacological and functional characteristics, including stimulation of adenylyl cyclase and appropriate raclioligand-binding activity. In this sense, there have been no surprises found with the cloning of this dopamine receptor subtype. However, it appears that there may be multiple D, receptors and that the molecular biology of this subtype is far from complete. Although some D1 receptors in both the rat striatum and kidney have recently been demonstrated to be independently linked to stlmulation of PI turnover rather than adenylyl cyclaae activityll*lz, the recently cloned D, receptor lacks the ability to stimulate t&e PI
signalling pathwap’. Moreover, the kidney also contains a D, receptor coupled to adenylyl cydase stimulation12 yet northern blot and polymerase chain reaction (PCR) analysis has failed to detect mRNA in this tissue for the cloned D1 receptor’8. There thus appear to be at least two other D, receptor genes remaining to be cloned and characterized. The rat Dz receptor cloned by Civelli and associates in 1988 was actually the first dopamine receptor subtype to be isolated and sequenced13. When expressed in mammalian cells, this receptor exhibits pharmacologically specific radioligand-binding activity and is functionally coupled to the inhibition of adenylyl q&se. The D2 receptor has subsequently been determined to exist as two protein &forms that differ in length by 29 amino acids and are derived from the same gene by alternative RNA splicinglcm. The location of the splice variation occurs within the third cyto$asmic loop, a region of the receptor believed to be involved in G protein coupling. Although this might suggest functional differences, both isoforms have been shown thus far to inhibit adenylyl cyclasels as well as to activate K+ channeW. The two receptor isoforms also appear to be pharmacologically identical’c’b. As there is no evidence yet to indicate that these receptors differ either functionally or pharmacologically, they have been designated by size: D2s, short and Dsl, long”. The mRNA distribution for these isoforms correlates well with dopaminergic projection areas with the highest levels being found in the caudate putamen, nucleus accumbens and olfactory tubercle. In all regions examined, the longer mRNA splice variant appears to represent the predominant isoform although the ratio of these forms can vary significantly*5*M. Interestingly, mRNA for both D2 receptor isoforms has also been found in the substantia nigra15Jnsuggesting an additional presynaptic or autoreceptor role for this cloned D2 subtype.
Cloning of a 4 receptor Although most brain regions known to exhibit Dz receptorbinding activity also express mRNA for this receptor subtype, some notable exceptions have been found. In particular, 4 receptors have been visualized by autoradiographic methods in the olfactory bulb, neocortex and paleocortex, as well as the hippocampus, but no corresponding mRNA could be detected in these regions, suggesting potential Dz receptor heterogeneity=. Some of these discrepancies may now be resolved by the recent cloning of a new D3 dopamine receptor that is highly related to the & subtype. Using probes derived from the Dz receptor sequence, Sokoloff et al. have isolated a novel receptor from rat cDNA and genomic libraries=. The cloned receptor protein is 446 residues in length and contains seven putative membrane-spanning domains (Fig. 1). The amino acid sequence of the 4 receptor is very similar to that of the Dz subtype, exhibiting a homology of 52% overall but up to 7540% when only the transmembrane regions, which may be involved in ligand recognition, are considered (Fig. I). The overall membrane topography of the DY receptor is also similar to that of the Dz receptor in exhibiting a relatively large third intracellular loop and a short C-terminus (Fig. 1). This is similar, in turn, to the a2-adrenoceptors which, like the D2 receptor, are linked to adenylyl cyclase inhibitionlo. Characterization of the gene for the 4 receptor also reveals multiple introns within the protein-coding region, three of which are in similar or identical locations to their pasi tions in the Dz receptor gene. This high homology with respect to both protein sequence and genomit organization suggests that the 4 and 4 receptor genes diverged from a common precursor gene in recent evolutionary history. The intron locations in the D3 gene suggest that alternative RNA splicing could not occur without a major disruption of receptor structure. Expression and binding studies in Chinese hamster ovary (CHO) cell9 indicate that the 4 receptor exhibits a pharmacology similar to yet distinct from that of the Dz receptor. In general, among the
TiPS - \anrr~y 1992/Vol. 721
EXll3ACELLlKAR
lNlRACELLUlAA
Fg. 1. Pmpowd mmrrbr8ne fopography of Un? ral & dopemine reCWor ad ifs hkmfam~~ers&hfl;~onuM relarionshiptilh!heDp~. basis d hy@xlhy tdysis Solid ctmks indicate amino acids that 811) identical h both fhe Dj and Q1 rs@ors CHO: polential N-W& gl)cosyfafkm sites.
ligands examined, antagonists appear to be more potent at the D2 than the D, receptor while the opposite is true for agonists. Among the antagonists, only the autoreceptor-selective putative agents A)76 and UH232 are more potent at the DI receptor,but only by three- to fourfold. Sokoloff et II/. have additionally noted that many of the ‘typical’neuroleptics (e.g. haloperidot), which have a high propensity to induce extrapyramidal side effects, are 10-20fold more potent at D2 than DJ receptors,whereas some ‘atypical’ neuroleptics(e.g. clozapine),which induce fewer extrapyramidal symptoms,are only two- to threefold D~-selective~X.This suggests that differential blockade of D2 and DX receptors may play an important role in antipsychotic therapy. Among all the ligands tested, the agonist quinpirole exhibits the greatest DJ selectivity, being about 100-fold more potent at D:, relative to I& receptors.The overall pharmacologicalsimilarity between the Dr and DJ receptors suggeststhat many of the effects of dopaminergic drugs on the CM that have been attributed to Da receptor stimulation or blockade may, in fact, be due to interactionsat the DJ receptor.
Thus far the signal transduction pathway associated with the Dq receptor has not been identified. When examined in transfected CHO cells, the Ds receptor was found to have no effect on adenylyl cyclase inhibition or stimulation. By contrast,the Dr receptor potently inhibited adenylyl cy clase activity in parallel CHO experiments. This observation is somewhat surprising in light of the structural similarity between the Da and DJ receptors.It should be noted, however, that binding of agonists to the expressed DJ receptor was found to be insensitive to guanine nucleotides, suggestingthe lack of appropriate C protein coupling in the CHO cells. Conceivably,the Da receptor could be linked to adenylyl cyclase inhibition, as is the Dz receptor, but through a different Gi protein that is not expressedin the CHO cells. Alternatively, the D1 receptor might be coupled to different signalling mechanisms, such as K’ and Ca?’ channel regulation, as has been suggestedfor the D2 receptor system. The distribution of the Da receptor mRNA was found to overlap partially with but differ significantly from that of the 4 receptoraa.Northern analysis indicated
the relative abundance of message was olfactory tubercle > hypothalamus > striatum > substantia nigra. In situ hybridization anaiysis confirmed this distribution and further showed the Da receptor mRNA to be predominantly located in the ventral striatum, nucleus accumbens and other ‘limbic’ areas such as the hippocampus, septum and mammiliary nuclei. This suggestsan important associationof the Da receptorwith cognitive and emotional CNS functions. Given this observation, as well as its demonstrated affinity for neuroieptics, it wil be interesting to perform linkage analysisfor the human Ds receptor gene in various schizophrenic kindred. The location of the Ds receptor in the substantia nigra also suggeststhat it may function as an autoreceptor and preliminary pharmacology is consistent with this possibility. Since the Da receptor is t!so located presynap tically, more work will be requirec to define the role of each receptor in regulating the activity and function of dopamine neurons. Doprmine receptor nomencI8hue The cloning of the receptor designated ‘D< by Sokolaff et aLaa brings up an important issue with respect to the nomenclature of dopamine receptorsas well as that of receptorsin general. Classically, receptor subtypes have been defined primarily on the basis of their pharmacological and, in some cases, functional properties. The advent of molecular cloning techniques has provided a complementary and perhaps more powerful method for receptor classification based on the sequence of the receptor proteins. Using this information, receptor subtypes can be grouped into families and subfamilies on the basis of their structural homology in addition to their pharmacology. An excellent example can be four&d using the adrenoceptor family for which many of the receptor subtypes have been cloned and sequenced. This family of receptors has recently been defined as consisting of ut-, - and @adrencrceptor subzmilies. Within each subfamily, the receptor subtypes exhibit 4050% overall homology with each other (e.g. (~1~vs ale or fit vs &) but up to 7040% homologyamong
TiPS -- january 1991/Vol. 121
the transmembrane regions. By contrast, if receptors between subfamilies (e.g. Qrs vs au) are compared, the transmembrane homology drops to -40%. The transmembrane regions appear to constitute the ligand-binding domains and thus ultimately dictate the receptor pharmacology. If this classification approach can be extrapolated to the dopamine receptor family, given their structural and pharmacological similarities, the D2 and D3 receptors are co-members of a distinct dopamine receptor subfamily. In my view, it would make sense to incorporate the nomenclature of the new Da receptor into the existing 4 subcategory. Thus, the first Ds receptor that was clonedra would be referred to as Daa (with Dz~.s and D2a.t. designating the short and long isoforms, respectively) with the Ds receptor being designated 4s. This might be especially helpful since many previous investigations of ‘4 receptom may have actually involved the Ds receptor. Adoption of this nomenclature would also provide a rational basis for the assignment of new dopamine receptors yet to be identified and cloned. Thus, on the basis of their structural and pharmacological profiles, novel dopamine receptor subtypes would either be placed into the existing Dr or Ds subcategories (using A, 8, C, etc. designations) or, if sufficiently divergent, would constitute prototypical membem of new (D3, D,, etc.) dopamine receptor subfamilies. DAVID R. SIBLEY Expcrimvlal ?7wrapc1rrinBrawrk, Nntiotd htifufe of NewoloRicd Disorders 14 Stroke, Bcthrsda,MD 20892. USA.
References 1 Kebrbian, J. W. and Caine, D. B. (1979) Nature 277,93-% 2 V*Lr, L. and Meldolesi, J. (1989) Trrmfs Phamlacof.Sn. 10.74-77 3 Soko)off, P., Martres. M. P. and Schwartz, J. C, (1980) Nn*rytr-SrBnliFIIcbrq’s Arch. Fharmmool. 315,89-102 4 Seeman, I? (1982) Biorhral. Phsrrtmcol. 31.2563-2568 5 Creese, I., Sibley, D. R., Hamblin, M. W. and Leff, 5. E. (1983) Armc. Rrv. NLWOxi. 6, 43-71 6 Monsma. F. 1.. Jr, Mahan, L. C., McVittie, L. D., Gerfen, C. R. and Sibley, D. R. (1990) Pmt. Natf Arad. Sri. USA 87.6723427 7 Dewy, A., Cingrich, J. A., hiardeau, P., Fremwu, R. T.. Jr, Bates, M. D. and Cemn, M. G. (1990) N~frw 347,72-76
9 8 Zhou, Q. Y. rf al. (1990) Natarc .347, 76-80 9 Sunahara, R. K. PI RI. (1990) Naterr 347, 80-83 10 O’Dowd, B. F., Letkowitz, R. J. and Camn, M. G. (1989) Atrnrc. Rrs. Nrlrrosci. 12.67-83 11 Mahan, L. C., Burch, R. M.. Monsma, F. J., Jr and Sibley. D. R. (1990) Pmr. Nat1 Arad. Sri. USA 87.2196-2200 12 FeMer, R. A., Felder, C..C.. Eisner, G. M. and lose, I’. A. (1989) Avr. 1. Physiof.275, F315-f327 13 Bunzow, J. R. cl RI. (1988) Natare 336, 783-787 14 Monsma, F. J., Jr, McVittie, L. D.. Gerfen, C. R., Mahan, L. C. and Sibley. D. R. (1989) Natrrc 342,926-929 15 Gims, B. et a/. (1989) Nnfarr342.923926 16 Grandy, D. K. c1 al. (1989) Pror. Nat/ Acud. Sri. USA 86.9762-9766 17 Selbie, L. A., Hayes, C. and Shine, J.
(1989) DNA 8,683-689 18 Dal Toso. R. CI al. (1989) EM60 /, 8, 4025-1034 19 Chio. C. L.. Hess. C. F., Graham. R. S. and Huff. R. M. (1990) Nnrrrrr 3.13. 266-269 20 O’Malley. K. L.. Mzck. F J . Ch&hn. K. Y. and Todd, ft. D. (1990) 610. Cl?OSiSfry 29, 1367-1371 21 Einhom. L. C., Falardeau. P., Camn, M. C. and Oxford. G. S. (1990) br. Nrtrrosd. Ahsfr. 16, 3B2 22 Mansour, A. cf al. (1990) /. Nrrtrosri. 10, 2587-26GlJ 23 Sokoloff, I’., Gims, 8.. Ma&es, hi. I’.. Bouthenet. M. L. and Schwartz. J. C. (1990) Nntrrrc347, 146-151 Aj76: cir-(+!-(ls,Za)-5 methoxy-l-methylZ-(n-pmpylamino) trtralin lJ= cis-(+)-(ls,2~)-5 methoxy-lmethyl-2-(di-s-pmpylamino) tetralin
Cloning and structur~function of the Ha histamine receptor There are a very limited number of mechanisms by which neurotransmitters and hormones can trigger an intracellular response on binding to their cell surface receptors. Each receptor mechanism and core structure has been well conserved during evolution; however, this is not so for binding site domains and other specific regions where evolutionary variability has ensured large numbers of receptors which belong to a specific gene family but are activated by different ligands. One such superfamily consists of the receptors that catalytically activate heterotrimeric GTPases (G proteins), which themselves
constitute another superfamily of proteins. It now seems likely that there are over 150 different Gprotein coupled receptors, of which -40 have been cloned. Many of these receptors have monoamines as their endogenous ligands e.g. noradrenaline, dopamine, acetylcholine and 5-HT, and have specific conserved residues which are thought to form part of the binding site. These residues are absent in the cloned peptide (e.g. neurokinin) or hormone (e.g. LH) receptors. An important new member of the monoamine receptor family, the H, histamine receptor, has now been cloned’ using a poty-
7
of a ‘Di receptor subtype expands dopamine receptor family
Cloning
Ten years ago, only two different dopamine receptor subtypes were definitively known to exist: the D, receptor, linked to the stimulation of adenylyl cyclase, and the D2 receptor which either inhibited adenylyl cyclase or was unlinked to this enzyme’. DZreceptors have now been suggested to be linked to additional second messenger systems, including inhibition of phosphatidylinositol (PI) tumover, activation of K+ channels and inhibition of Ca2+ channel activityz. The existence of additional DS and Dd receptors was proposed in the early 19&W4, but this was based on differing agonist affinities in ligand-binding assays and subsequently shown to represent different affinity states of the known Dt and D2 receptorss. New information derived from cloning of the D1 and D2 receptors, as well as that of a putative D3 subtype, now indicates that the dopamine receptor family could be even more heterogeneous than previously suspected.
The D1 receptor linked to the activation of adenylyl cyclase has recently been cloned from both rat and human tissues-. This receptor is 446 amino acids in length and has aeven putative membranespanning domains typical of the G protein-linked receptor familylo. When expressed in mammalian cells, the cloned D1 receptor exhibits the expected pharmacological and functional characteristics, including stimulation of adenylyl cyclase and appropriate raclioligand-binding activity. In this sense, there have been no surprises found with the cloning of this dopamine receptor subtype. However, it appears that there may be multiple D, receptors and that the molecular biology of this subtype is far from complete. Although some D1 receptors in both the rat striatum and kidney have recently been demonstrated to be independently linked to stlmulation of PI turnover rather than adenylyl cyclaae activityll*lz, the recently cloned D, receptor lacks the ability to stimulate t&e PI
signalling pathwap’. Moreover, the kidney also contains a D, receptor coupled to adenylyl cydase stimulation12 yet northern blot and polymerase chain reaction (PCR) analysis has failed to detect mRNA in this tissue for the cloned D1 receptor’8. There thus appear to be at least two other D, receptor genes remaining to be cloned and characterized. The rat Dz receptor cloned by Civelli and associates in 1988 was actually the first dopamine receptor subtype to be isolated and sequenced13. When expressed in mammalian cells, this receptor exhibits pharmacologically specific radioligand-binding activity and is functionally coupled to the inhibition of adenylyl q&se. The D2 receptor has subsequently been determined to exist as two protein &forms that differ in length by 29 amino acids and are derived from the same gene by alternative RNA splicinglcm. The location of the splice variation occurs within the third cyto$asmic loop, a region of the receptor believed to be involved in G protein coupling. Although this might suggest functional differences, both isoforms have been shown thus far to inhibit adenylyl cyclasels as well as to activate K+ channeW. The two receptor isoforms also appear to be pharmacologically identical’c’b. As there is no evidence yet to indicate that these receptors differ either functionally or pharmacologically, they have been designated by size: D2s, short and Dsl, long”. The mRNA distribution for these isoforms correlates well with dopaminergic projection areas with the highest levels being found in the caudate putamen, nucleus accumbens and olfactory tubercle. In all regions examined, the longer mRNA splice variant appears to represent the predominant isoform although the ratio of these forms can vary significantly*5*M. Interestingly, mRNA for both D2 receptor isoforms has also been found in the substantia nigra15Jnsuggesting an additional presynaptic or autoreceptor role for this cloned D2 subtype.
Cloning of a 4 receptor Although most brain regions known to exhibit Dz receptorbinding activity also express mRNA for this receptor subtype, some notable exceptions have been found. In particular, 4 receptors have been visualized by autoradiographic methods in the olfactory bulb, neocortex and paleocortex, as well as the hippocampus, but no corresponding mRNA could be detected in these regions, suggesting potential Dz receptor heterogeneity=. Some of these discrepancies may now be resolved by the recent cloning of a new D3 dopamine receptor that is highly related to the & subtype. Using probes derived from the Dz receptor sequence, Sokoloff et al. have isolated a novel receptor from rat cDNA and genomic libraries=. The cloned receptor protein is 446 residues in length and contains seven putative membrane-spanning domains (Fig. 1). The amino acid sequence of the 4 receptor is very similar to that of the Dz subtype, exhibiting a homology of 52% overall but up to 7540% when only the transmembrane regions, which may be involved in ligand recognition, are considered (Fig. I). The overall membrane topography of the DY receptor is also similar to that of the Dz receptor in exhibiting a relatively large third intracellular loop and a short C-terminus (Fig. 1). This is similar, in turn, to the a2-adrenoceptors which, like the D2 receptor, are linked to adenylyl cyclase inhibitionlo. Characterization of the gene for the 4 receptor also reveals multiple introns within the protein-coding region, three of which are in similar or identical locations to their pasi tions in the Dz receptor gene. This high homology with respect to both protein sequence and genomit organization suggests that the 4 and 4 receptor genes diverged from a common precursor gene in recent evolutionary history. The intron locations in the D3 gene suggest that alternative RNA splicing could not occur without a major disruption of receptor structure. Expression and binding studies in Chinese hamster ovary (CHO) cell9 indicate that the 4 receptor exhibits a pharmacology similar to yet distinct from that of the Dz receptor. In general, among the
TiPS - \anrr~y 1992/Vol. 721
EXll3ACELLlKAR
lNlRACELLUlAA
Fg. 1. Pmpowd mmrrbr8ne fopography of Un? ral & dopemine reCWor ad ifs hkmfam~~ers&hfl;~onuM relarionshiptilh!heDp~. basis d hy@xlhy tdysis Solid ctmks indicate amino acids that 811) identical h both fhe Dj and Q1 rs@ors CHO: polential N-W& gl)cosyfafkm sites.
ligands examined, antagonists appear to be more potent at the D2 than the D, receptor while the opposite is true for agonists. Among the antagonists, only the autoreceptor-selective putative agents A)76 and UH232 are more potent at the DI receptor,but only by three- to fourfold. Sokoloff et II/. have additionally noted that many of the ‘typical’neuroleptics (e.g. haloperidot), which have a high propensity to induce extrapyramidal side effects, are 10-20fold more potent at D2 than DJ receptors,whereas some ‘atypical’ neuroleptics(e.g. clozapine),which induce fewer extrapyramidal symptoms,are only two- to threefold D~-selective~X.This suggests that differential blockade of D2 and DX receptors may play an important role in antipsychotic therapy. Among all the ligands tested, the agonist quinpirole exhibits the greatest DJ selectivity, being about 100-fold more potent at D:, relative to I& receptors.The overall pharmacologicalsimilarity between the Dr and DJ receptors suggeststhat many of the effects of dopaminergic drugs on the CM that have been attributed to Da receptor stimulation or blockade may, in fact, be due to interactionsat the DJ receptor.
Thus far the signal transduction pathway associated with the Dq receptor has not been identified. When examined in transfected CHO cells, the Ds receptor was found to have no effect on adenylyl cyclase inhibition or stimulation. By contrast,the Dr receptor potently inhibited adenylyl cy clase activity in parallel CHO experiments. This observation is somewhat surprising in light of the structural similarity between the Da and DJ receptors.It should be noted, however, that binding of agonists to the expressed DJ receptor was found to be insensitive to guanine nucleotides, suggestingthe lack of appropriate C protein coupling in the CHO cells. Conceivably,the Da receptor could be linked to adenylyl cyclase inhibition, as is the Dz receptor, but through a different Gi protein that is not expressedin the CHO cells. Alternatively, the D1 receptor might be coupled to different signalling mechanisms, such as K’ and Ca?’ channel regulation, as has been suggestedfor the D2 receptor system. The distribution of the Da receptor mRNA was found to overlap partially with but differ significantly from that of the 4 receptoraa.Northern analysis indicated
the relative abundance of message was olfactory tubercle > hypothalamus > striatum > substantia nigra. In situ hybridization anaiysis confirmed this distribution and further showed the Da receptor mRNA to be predominantly located in the ventral striatum, nucleus accumbens and other ‘limbic’ areas such as the hippocampus, septum and mammiliary nuclei. This suggestsan important associationof the Da receptorwith cognitive and emotional CNS functions. Given this observation, as well as its demonstrated affinity for neuroieptics, it wil be interesting to perform linkage analysisfor the human Ds receptor gene in various schizophrenic kindred. The location of the Ds receptor in the substantia nigra also suggeststhat it may function as an autoreceptor and preliminary pharmacology is consistent with this possibility. Since the Da receptor is t!so located presynap tically, more work will be requirec to define the role of each receptor in regulating the activity and function of dopamine neurons. Doprmine receptor nomencI8hue The cloning of the receptor designated ‘D< by Sokolaff et aLaa brings up an important issue with respect to the nomenclature of dopamine receptorsas well as that of receptorsin general. Classically, receptor subtypes have been defined primarily on the basis of their pharmacological and, in some cases, functional properties. The advent of molecular cloning techniques has provided a complementary and perhaps more powerful method for receptor classification based on the sequence of the receptor proteins. Using this information, receptor subtypes can be grouped into families and subfamilies on the basis of their structural homology in addition to their pharmacology. An excellent example can be four&d using the adrenoceptor family for which many of the receptor subtypes have been cloned and sequenced. This family of receptors has recently been defined as consisting of ut-, - and @adrencrceptor subzmilies. Within each subfamily, the receptor subtypes exhibit 4050% overall homology with each other (e.g. (~1~vs ale or fit vs &) but up to 7040% homologyamong
TiPS -- january 1991/Vol. 121
the transmembrane regions. By contrast, if receptors between subfamilies (e.g. Qrs vs au) are compared, the transmembrane homology drops to -40%. The transmembrane regions appear to constitute the ligand-binding domains and thus ultimately dictate the receptor pharmacology. If this classification approach can be extrapolated to the dopamine receptor family, given their structural and pharmacological similarities, the D2 and D3 receptors are co-members of a distinct dopamine receptor subfamily. In my view, it would make sense to incorporate the nomenclature of the new Da receptor into the existing 4 subcategory. Thus, the first Ds receptor that was clonedra would be referred to as Daa (with Dz~.s and D2a.t. designating the short and long isoforms, respectively) with the Ds receptor being designated 4s. This might be especially helpful since many previous investigations of ‘4 receptom may have actually involved the Ds receptor. Adoption of this nomenclature would also provide a rational basis for the assignment of new dopamine receptors yet to be identified and cloned. Thus, on the basis of their structural and pharmacological profiles, novel dopamine receptor subtypes would either be placed into the existing Dr or Ds subcategories (using A, 8, C, etc. designations) or, if sufficiently divergent, would constitute prototypical membem of new (D3, D,, etc.) dopamine receptor subfamilies. DAVID R. SIBLEY Expcrimvlal ?7wrapc1rrinBrawrk, Nntiotd htifufe of NewoloRicd Disorders 14 Stroke, Bcthrsda,MD 20892. USA.
References 1 Kebrbian, J. W. and Caine, D. B. (1979) Nature 277,93-% 2 V*Lr, L. and Meldolesi, J. (1989) Trrmfs Phamlacof.Sn. 10.74-77 3 Soko)off, P., Martres. M. P. and Schwartz, J. C, (1980) Nn*rytr-SrBnliFIIcbrq’s Arch. Fharmmool. 315,89-102 4 Seeman, I? (1982) Biorhral. Phsrrtmcol. 31.2563-2568 5 Creese, I., Sibley, D. R., Hamblin, M. W. and Leff, 5. E. (1983) Armc. Rrv. NLWOxi. 6, 43-71 6 Monsma. F. 1.. Jr, Mahan, L. C., McVittie, L. D., Gerfen, C. R. and Sibley, D. R. (1990) Pmt. Natf Arad. Sri. USA 87.6723427 7 Dewy, A., Cingrich, J. A., hiardeau, P., Fremwu, R. T.. Jr, Bates, M. D. and Cemn, M. G. (1990) N~frw 347,72-76
9 8 Zhou, Q. Y. rf al. (1990) Natarc .347, 76-80 9 Sunahara, R. K. PI RI. (1990) Naterr 347, 80-83 10 O’Dowd, B. F., Letkowitz, R. J. and Camn, M. G. (1989) Atrnrc. Rrs. Nrlrrosci. 12.67-83 11 Mahan, L. C., Burch, R. M.. Monsma, F. J., Jr and Sibley. D. R. (1990) Pmr. Nat1 Arad. Sri. USA 87.2196-2200 12 FeMer, R. A., Felder, C..C.. Eisner, G. M. and lose, I’. A. (1989) Avr. 1. Physiof.275, F315-f327 13 Bunzow, J. R. cl RI. (1988) Natare 336, 783-787 14 Monsma, F. J., Jr, McVittie, L. D.. Gerfen, C. R., Mahan, L. C. and Sibley. D. R. (1989) Natrrc 342,926-929 15 Gims, B. et a/. (1989) Nnfarr342.923926 16 Grandy, D. K. c1 al. (1989) Pror. Nat/ Acud. Sri. USA 86.9762-9766 17 Selbie, L. A., Hayes, C. and Shine, J.
(1989) DNA 8,683-689 18 Dal Toso. R. CI al. (1989) EM60 /, 8, 4025-1034 19 Chio. C. L.. Hess. C. F., Graham. R. S. and Huff. R. M. (1990) Nnrrrrr 3.13. 266-269 20 O’Malley. K. L.. Mzck. F J . Ch&hn. K. Y. and Todd, ft. D. (1990) 610. Cl?OSiSfry 29, 1367-1371 21 Einhom. L. C., Falardeau. P., Camn, M. C. and Oxford. G. S. (1990) br. Nrtrrosd. Ahsfr. 16, 3B2 22 Mansour, A. cf al. (1990) /. Nrrtrosri. 10, 2587-26GlJ 23 Sokoloff, I’., Gims, 8.. Ma&es, hi. I’.. Bouthenet. M. L. and Schwartz. J. C. (1990) Nntrrrc347, 146-151 Aj76: cir-(+!-(ls,Za)-5 methoxy-l-methylZ-(n-pmpylamino) trtralin lJ= cis-(+)-(ls,2~)-5 methoxy-lmethyl-2-(di-s-pmpylamino) tetralin
Cloning and structur~function of the Ha histamine receptor There are a very limited number of mechanisms by which neurotransmitters and hormones can trigger an intracellular response on binding to their cell surface receptors. Each receptor mechanism and core structure has been well conserved during evolution; however, this is not so for binding site domains and other specific regions where evolutionary variability has ensured large numbers of receptors which belong to a specific gene family but are activated by different ligands. One such superfamily consists of the receptors that catalytically activate heterotrimeric GTPases (G proteins), which themselves
constitute another superfamily of proteins. It now seems likely that there are over 150 different Gprotein coupled receptors, of which -40 have been cloned. Many of these receptors have monoamines as their endogenous ligands e.g. noradrenaline, dopamine, acetylcholine and 5-HT, and have specific conserved residues which are thought to form part of the binding site. These residues are absent in the cloned peptide (e.g. neurokinin) or hormone (e.g. LH) receptors. An important new member of the monoamine receptor family, the H, histamine receptor, has now been cloned’ using a poty-