Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
JOURNAL OF RECEPTOR RESEARCH, 11(1-4), 521-534 (1991)
MOLECULAR CHARACTERIZATION OF G-PROTEIN COUPLED RECEPTORS: ISOLATION AND CLONING OF A D1 DOPAMINE RECEPTOR
Jay A. Gingrich, Allen Dearry, Pierre Falardeau, Robert T. Fremeau Jr., Michael D. Bates, and Marc G. Caron Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, N.C.
ABSTRACT
This article summarizes the recent progress our laboratory has made in understanding the molecular characteristics of the D1 dopamine receptor. The D1 dopamine receptor from rat striaturn has been purified to near homogeneity using a combination of several chromatographic steps. Furthermore, the gene for the human D1 dopamine receptor has been cloned, sequenced, and expressed. The cloned receptor has all the pharmacologic and biochemical properties of the classical D1 receptor coupled to adenylyl cyclase which has been previously described in the central nervous system.
INTRODUCTION G-protein-coupledreceptors represent a large family of membrane proteins involved in mediating the effects of a large member of hormones and neurotransmitters. Binding of a hormone to one of these receptors promotes the interaction of the ligandreceptor complex with and activation of a heterotrimericG-protein which in turn activates an effector system such as adenylyl cyclase, phospholipase C or an ion channel (1,Z). The biochemical processes by which this signal transduction occurs are fairly well understood. Insights into these processes have been facilitated recently by the elucidation of the primary sequence of the major components of these systems via the isolation and cloning of the genes and cDNA's for these proteins (1 2 ) .
521 Copyright
0 1991 by
Marcel Dekker, Inc.
522
GINGRICH ET A L . The approach initially taken to elucidate the structure of G-protein coupled
receptors such as the adrenergic receptors, was to purify these receptors in sufficient quantities to allow the determinationof partial amino acid sequence (3,4).Based on Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
peptide sequence, oligonucleotide probes were synthesized and cDNA and genes were isolated by recombinant DNA techniques. The first of the receptors to be cloned was the hamster p2 adrenergic receptor (5).Soon after, several other receptors were also cloned such as the pl adrenergic receptor subtype, and several rnuscarinic receptor These proteins all share in common the same seven transrnembrane subtypes (6-9). domain arrangement (4).Within these transrnembrane domains, these proteins display considerable amino acid identity, varying from almost 75% within subtypes such as pl and p2 adrenergic receptors to 30-35% when comparing distant receptors such as p adrenergic and muscarinic receptors (10). This extensive sequence conservation has been used as a strategy in the cloning of several related members of this family of proteins (1,11,12). The cDNAs and/or genes for a large number of G-protein coupled receptors have been isolated thus far. Whereas all of these receptors appear to be very similar in their structural properties, an unexpected finding has been the large number of previously unsuspected receptor subtypes revealed by the molecular biology approach.
For example, previously, only four different adrenergic receptors had been distinguished by pharmacologicaland biochemical studies. It is now evident from the isolation of cDNAs and genes, that at least 9 adrenergic receptors exist, three p subtypes, three a1 subtypes, and three a2 subtypes (1,13-16).The exact physiologicalrole that these various receptor subtypes play remains to be examined. DOPAMINE RECEPTORS. In the 25 years since dopamine was identified as a
neurotransmitterin the CNS, great progress has been made towards understandingthe role of dopaminergic systems in normal and pathologic states. The effects of dopamine are presumably mediated by two distinct G-protein coupled receptors: D1 receptors that are coupled to stimulation of adenylyl cyclase and D2 receptors that inhibit the enzyme but also activate K+ channels (17). However, in contrast to the wealth of studies describing the functional effects of dopamine, insights into the biochemistry of the receptors which mediate these effects have lagged far behind. This generalization is especially true for the D1 dopamine receptor. Although the first dopamine-stimulated activation of adenylyl cyclase was described in 1971 (18), very little progress has been
G-PROTEIN COUPLED RECEPTORS
523
made towards understanding the biochemistry of this receptor. Beginning 7 or 8 years ago our group initiated an approach to elucidate the molecular events underlying the effects of dopamine acting through D1 and D2 dopamine receptors. Our specific Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
interest was in understanding the molecular properties of these receptor proteins, and in this brief report, we review some of the progress towards this goal which our group has made with the D1 receptor subtype.
RESULTS IDENTIFICATION AND PURIFICATION OF THE D7 DOPAMINE RECEPTOR.
In
collaboration with scientists at the Schering-PloughCorporation, we sought to develop a D1 selective benzazepine antagonist which could be used as a photoaffinity probe for this receptor. The result of this collaboration was the compound, [1251]SCH39582, which could be photo-crosslinked to the D1 receptor by the bifunctional reagent, SANPAH (19). Using this technique, the D1 receptor was identified to be a 72 kDa glycoprotein (see Figure 1A) and from work with the 82 adrenergic receptor (20), it was expected that the ligand binding subunit identified by this photoprobe would also possess the adenylyl cyclase coupling capacity of this receptor as well. One of the most powerful tools in biochemistry has proven to be the ability to study the properties of a pure protein in in vitro assays. For this reason we undertook the purification of the D1 receptor. With the development of a D1 selective antagonists and which could photo-crosslink the D1 receptor, we believed that these compounds could be adapted for use as biospecific affinity chromatography ligands. For this purpose, SCH39111 was synthesized. When immobilized to Sepharose beads through an appropriate spacer arm, SCH39111 was able to function as a biospecific affinity ligand capable of 200-300 fold purification of the receptor from crude digitonin-solubilizedrat striatal membranes (21). Further purification (Figure 1B) of this partially purified receptor using ion exchange, size exclusion, and lection chromatographyyielded a preparation with a specific activity of ca. 11,000 pmol [3H]SCH23390boundhng protein (theoretical specific activity being approximately 13,000 pmollmg). When this preparation was radioiodinatedusing Bolton-Hunterreagent, a predominant band at 72 kDa was observed after SDS-PAGE and autoradiography (Figure 1C). Thus, using this
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 24
GINGRICH ET A L .
FIGURE 1. Identification and purification of the rat striatal D1 dopamine receptor. A. A n autoradiogram of an SDS-PAGE of D1 receptor crosslinkedto [1251)SCH39582 using the bifunctional reagent. SANPAH. In the absence (-) and presence (+) of 1 fl (+) butachfml. The radoligand is imrporated into a protein wdh an apparent mass of 72 kDa B. An Outline of the procedure used to obtain highly puriiied D1 receptor. C. An autoradiogram of an SDS-PAGE aiter treating the purified receptor preparation(specific activily = ca. 11,000 pmol [3H]SCH23390binding siteslrng protein) wth [1251]Bolton-Hunterreagent. Note that the predominant protein in this preparationis the 72 kDa band which wmgrates with photoatfinity labelled receptor.
procedure, we were successful in obtaining highly purified preparations of the D1 receptor. CLONING
OF THE GENE FOR THE D1 DOPAMINE RECEPTOR. As summarized in
the introduction, the application of molecular biology to receptor research has proven extremely fruitful in the last several years. An early hope during the course of these studies was that purification might provide enough receptor protein to allow partial peptide sequence analysis of this receptor. However, given the difficulty in obtaining
G - P R O T E I N COUPLED RECEPTORS
525
the large amounts of pure receptor protein necessary for this purpose, an alternate strategy was employed to isolate the gene and cDNA for the D1 receptor. Taking advantage of the sequence information recently described for the D2 dopamine Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
receptor (12), we designed an oligonucleotide probe based on its putative second transmembrane domain and used this probe to screen a human retina cDNA library. The second transmembrane domain was chosen because it is highly conserved between other receptor subtypes and was thus thought likely to be conserved between the D2 and D1 receptors. A retinal library was chosen because dopamine is the predominant catecholaminein this tissue. This screening yielded several D2 clones as well a unique clone (D233) which upon sequencing, appeared to encode a novel member of the Gprotein coupled receptor family. The clone D233 was not full length, apparently lacking the amino terminus and the first transmembrane domain. A full length clone (HGL26) was obtained after screening a human genomic library with a 700 bp Pvu II fragment of D233. Sequencing of HGL26 confirmed its identity with D233, and also demonstrated that the gene for this novel receptor was intronless within its coding region (a result predicted from genomic Southern analysis). The deduced amino acid sequence and the predicted transmembrane topology of the open reading frame of HGL26 are shown in Figure 2 (22). Several features of this receptor sequence are of note. First, this receptor fits the well established pattern of seven hydrophobic segments (thought to be membrane spanning) which are found in all known G-protein coupled receptors. The protein also possesses 2 consensus sequences for N-linked glycosylation on its proposed extracellular domains. Furthermore, several consensus sites for protein kinases A and C as well as potential sites for agonist dependent receptor kinases such as the p adrenergic receptor kinase (23) are found in the proposed intracellular domains. That this receptor might be a catecholamine receptor seemed likely since this protein possessed the aspartate and two serine residues thought to be involved in catecholamine binding (24). However, the identification of this gene as the human D1 receptor would depend on analysis of the expressed protein. EXPRESSION OF THE CLONED D1 RECEPTOR GENE. To study the properties of
the protein encoded by HGL26, the coding region was subcloned into the mammalian expression vector pCMV5 in both a sense (pCMV5IDl) and antisense (pCMV5/Dl') orientation and used to transfect monkey kidney cells (COS7) cells in a transient fashion.
transmembrane domains are those traversing the stippled gray bar (plasma membrane). Potential sites of N-linked glycosylation are indicated by the "Y"-shaped structures made of small circles. Consensus sites for CAMP dependent protein kinase (A) and protein kinase C (C) phosphorylation are shown by the darkened circles.
FIGURE 2. Primary sequence and putative transmembrane topolgy of the human D1 dopamine receptor. The primary sequence is represented by single letter codes for each amino acid. The predicted
In tracellular
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
U
r
d
n
2
n
n
G-PROTEIN COUPLED RECEPTORS
527
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
TABLE I Antaaonists;
!wm!!a
SCH23390 cis pifluthixol (+) butaclamol cis flupenthixol SCH23388 ketanserin spiperone prazosin (-) butaclamol alprenoloI raclopride
0.1 1 0.20 0.90 1.6 22 190 220 3,000 4,900 16,000 >72,000
Aaonlsts: 20 fenoldopam SK F38393 87 210 (-)apomorphine 2,500 dopamine quinpirole 14,000 serotonin >15,000 epinephrine >55,000
Ligand Binding Pharmacology of the Cloned D1 Dopamine Receptor Expressed In COS-7 Cells. Cos-7 cells were transfected as described (22). Membranes prepared from these cells display saturable high affinity binding sites for [1251]SCH23982(0.35 nM, n=6). Various compounds were tested for their potency in competition assays with [1251]SCH23982. The results are expressed here as the average of at least three determinations. Agonists did not display biphasic curves in crude membrane preparations.
Figure 3 shows the result obtained when several Vitiated ligands specific for various types of biogenic amine receptors were tested on membranes from transfected cells. As shown, [3H]SCH23390 binding sites were increased ca. 20-fold on cells transfected with the sense construct compared with the antisense control. The binding sites for other ligands were not significantly increased, suggesting that HGL26 might be encoding the D1 dopamine receptor. Supporting this hypothesis, the binding of I3H]SCH23390 and its related compound [1251]SCH23982was found to be saturable and of high affinity (0.10 and 0.35 nM respectively). Furthermore,when several compounds were tested
for their ability to compete for [1251]SCH23982 binding to the expressed receptor, the order of potency observed in these experiments was entirely consistent with the pharmacology of the D l receptor from the caudate and putamen (25). The observed dissociation constants (Kds) for several of these compounds are summarized in Table I. Thus on the basis of ligand binding properties alone, the identity of this cloned receptor can be safely secured as the D1 dopamine receptor.
GINGRICH ET A L .
525
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
6000
5000
'
E 3000 Q.E 0-
1000
C
SCH SPIRO KET
YOH
ALP
FIGURE 3. Identificationof HGL26 protein product as the D1 dopamine receptor using ligand binding. Cells (cos-7) were transfected transiently with either the sense (pCMWD1) or antisense (pCMWD1') constructs and membrane homogenates were tested for tritiated ligand (1 nM +/- 10 pM competitor) binding 48 hr later. As shown cells transfected with the sense construct bound 18-fold more tritiated SCH23390 than cells transfected with the antisense construct . In contrast, the binding of other tritiated antagoniststested was similar in each transfection condition. SCH; SCH 23390 competed with cis flupenthixol, SPIRO; spiroperidol competed with cis flupenthixol, KET; ketanserin competed with serotonin, YOH; yohimbine competed with epinephrine, ALP; alprenolol competed with epinephrine.
G-PROTEIN COUPLED RECEPTORS
529
TABLE II
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
Conditions: basal dopamine dopamine+SCH23390 doparnine+raclopride dopamine+propranolol SKF38393 quinpirole
adenylyl cyclase actlvlty fold stlmulatlon (pmollmg protein 60 min) 235*5 1079k55 3 19+24 1128f129 1049k43 605k49 285+8
1 .o 4.6 1.4 4.8 4.5 2.6 1.2
Signal Transductlon of the Cloned D1 Receptor Expressed in Mouse L(TK) cells. L(Tk-) cells were cotransfectedwith pCMV5ID1 and a neomycin resistance plasmid. Clonal cells were selected which possessed G418 resistance. Membranes from these cells were assayed for dopamine stimulated adenylyl cyclase activity by previously described methods using maximally effective doses of agonist dopamine = 100 FM ; all other compounds were present at 1 bM). Notice that KF38393 is only a partial agonist at the D1 dopamine receptor for adenylyl cyclase stimulation which is in accord with previous results with this compound.
L
To assess the second messenger system of this cloned D1 dopamine receptor,
pCMVUD1 was used to permanently drive the expression of the D1 receptor in mouse L(tk-)cells, a host normally devoid of this receptor. Clonal cell lines were isolated which expressed 50-500 fmol receptorhng protein and these cells were then tested for their ability to stimulate the adenylyl cyclase second messenger system. Table II demonstrates that cells expressing this receptor are able to stimulate adenylyl cyclase activity 5-fold over basal and that this activity has all the pharmacologic properties expected of the D1 dopamine receptor. Importantly, no dopamine stimulated adenylyl cyclase activity can be elicited in untransfectedcells.
LOCALIZATION OF THE MESSAGE FOR THE DI DOPAMIME RECEPTOR.
Several techniques such as northern blot analysis, in situ hybridization, and polymerase chain reaction (PCR) were used to detect the messenger RNA for the D1 receptor in various peripheral organs and brain regions. All of these methods in concert yield a consistent picture of receptor expression. Message for this recept has the highest expression in the caudate nucleus, nucleus accumbens, and olfactory tubercle-- with
GINGRICH ET A L .
5 30
frontal cortex and hippocampus showing somewhat lower levels. Interestingly, two tissues which have relatively high levels of D1 dopamine receptors have undetectable levels of receptor message: substantia nigra and kidney. In the case of the substantia Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
nigra, this finding is consistent with the previously held hypothesis developed from lesioning studies (26) that D l dopamine receptors in this tissue exist on presynaptic nerve terminals whose cell bodies originate in the caudate. In the other instance, the kidney is known to possess postsynaptic D1 dopamine receptors, and thus the failure to find receptor message in this tissue (using the sensitive technique of PCR amplification as well as traditional northern blot analysis) suggests that this tissue may well contain a distinct but closely related subtype of the D1 receptor. This hypothesis is consistent with numerous pharmacologic discrepancies which have been noted between central and peripheral D1 dopamine receptors
DISCUSSION In summary, over the last several years our laboratory has progressedfrom our early attempts to learn more about the molecular characteristicsof the D1 receptor through photoaffinity labelling and purification of this receptor to elucidation of its deduced primary sequence and gene structure using molecular biological techniques. The tools are now in place to begin the process of studying the molecular aspects of signal transduction and regulation of this receptor. As summarized in this paper the human D1 dopamine receptor characterized here has all the properties of the classical D1 dopamine receptor characterized in the striaturn of several species (27,28). This receptor is typical of other G-protein coupled receptors. Overall, the D1 dopamine receptor sequence is most similar to the p l adrenergic receptor closely followed by the 02 dopamine and p2 adrenergic receptors. This emphasizes the fact that although D1 and D2 receptors both bind dopamine, they are quite different at the biochemical level. This is further highlighted by the fact that the D1 receptor appears to be intronless within its coding region, whereas the 0 2 receptor has several introns (29). One of the more interesting aspects of this work is the evidence which points to the possible presence of other subtypes of D1 dopamine receptors. The first evidence as mentioned above is the incongruence between message and receptor binding sites as observed in the kidney. A second line of evidence comes from the signal
G-PROTEIN COUPLED RECEPTORS
531
transduction studies. In mouse L(tk-) cells expressing the D i receptor, dopamine promotes stimulation of adenylyl cyclase or accumulation of intracellular CAMP but does not elicit stimulation of phosphatidylinositol hydrolysis (22). However, there is evidence Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
both from studies with striatal and kidney preparations (30-32) that dopamine through a D1 receptor can stimulate accumulation of diacylglycerol and inositol trisphosphate
under certain conditions. This suggests that a D1 receptor distinct from cloned brain receptor may exist that is coupled selectively to the phosphatidylinositol hydrolysis pathway. Thus, even at this early stage of characterizationof this receptor, evidence suggests that this receptor may be part of a larger family of D1 dopamine receptors. These findings are consistent with several other pharmacological, biochemical, electrophysiological and behavioral considerations (17). The tools are now in place to attempt to shed light on this heterogeneity and to study the molecular aspects of signal transduction through this receptor as well as the regulation of its responsiveness that may be a key element of its role in certain disorders.
ACKNOWLEDGEMENTS We thank V. Clack and N. Godinot for technical assistance. This work was supported in part by NIH grant NS19576. J.A.G. and M.D.B. are both fellows in the Medical Scientist Training Program.
REFERENCES 1.
Lefkowitz, R.J.; and Caron, M.G. Adrenergic receptors: Models for the study of receptors coupled to guanine nucleotide regulatory proteins. J. Biol. Chem. 263, 4993-4996, 1988.
2.
Casey, P.J.; and Gilman, A.G. G-protein involvement in receptor-effector coupling . Ann. Rev. Biochem. 56, 2577-2581, 1988.
3.
Benovic, J.L.; Shorr, R.G.L.; Caron, M.G.; and Lefkowitz, R.J. The mammalian p2 adrenergic receptor: purification and characterization. Biochemistry 23, 4510-4518, 1984.
4.
Dohlman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry 26, 2657-2664, 1987.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 32
GINGRICH ET A L .
5.
Dixon, R.A.F.; Kobilka, B.K.; Strader, D.J. ; Benovic, J.L.; Dohlman, H.G.; Frielle, T.; Bolanowski, M.A.; Bennett, C.D.; Rands, E.; Diehl, R.E.; Mumford, R.A.; Slater, E.E.; Sigal, IS.; Caron, M.G.; Lefkowitz, R.J.; and Strader, C.D. Cloning of the gene and cDNA for mammalian P-adrenergic receptor and homology to rhodopsin. Nature 321, 75-79, 1986.
6.
Yarden, Y.; Rodriguez, H.; Wong, S.K.-F.; Brandt, D.R.; May, D.C.; Bumier, J.; Harkins, R.N.; Chen, E.Y.; Ramachandran, J.; Ullrich, A,: and Ross, E.M. The avian P-adrenergic receptor: Primary structure and membrane topology. Proc. Natl. Acad. Sci. USA 83, 6795-6799, 1986.
7.
Kub,T.; Fukuda, K.; Mikami, A,; Maeda, A.; Takahashi, H.; Mishina, M.; Haga, T.; Haga, k.; Ichiyama, A.; Kangawa, K.; Kojima, M.; Matsuo, H.; Hirose, T.; and Numa, S. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323, 411-416, 1986.
8.
Kubo,T.; Maeda, A.; Sugimoto, K.; Akiba, 1.; Mikami, A.; Takahashi, H.; Haga, T.; Haga, K.; Ichiyama, A.; Kangawa, K.; Matsuo, H.; Hirose, T.; and Numa, S. Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence. FEBS. Lett. 209, 367-373, 1986.
9.
Bonner, T.I.; Buckley, N.J.; Young, A.C.; and Brann, M.R. Identificationof a family of muscarinic acetylcholine receptor genes. Science 237, 527-532, 1987.
10.
ODowd, B.F.; Caron, M.G.; and Lefkowitz, R.J. Structure of the adrenergic and related receptors. Ann. Rev. Neurosci. 12, 67-83, 1989.
11.
Kobilka, B.K.; Frielle, T.; Collins, S.; Yang-Feng, T.; Kobilka, T.S.; Franke, U.; Lefkowitz, R.J.; and Caron, M.G. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329, 75-79, 1987.
12.
Bunzow, J.R.; VanTol, H.H.M.; Grandy, D.K.; Albert, P.; Salon, J.; Christie, M.; Machida, C.A.; Neve, K.A.; and Civelli, 0. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature, 336, 783-787, 1988.
13.
Emorine, L.J.; Marullo, S . ; Briend-Sutren,M.M.; Patey, G.; Tate, K.; DelavierKlutchko, C.; and Strosberg, A.D. Molecular characterizationof the human beta 3-adrenergic receptor. Science 245, 1118-1121, 1989.
14.
Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; and Kobilka, B.K. Cloning and expressionof a human kidney cDNA for an a2 adrenergic receptor subtype. Proc. Natl. Acad. Sci USA 85, 6301-6305, 1988.
15.
Lomasney, J.W.; Lorenz, W.; Allen, L.F.; King, K.; Regan, J.W.; Yang-Feng, T.L.; Caron, M.G.; and Lefkowitz, R.J. Expansion of the a2-adrenergic receptor family: Cloning and characterizationof a human a2 receptor subtype, the gene for which is located on chromosome 2. Proc. Natl. Acad. Sci. USA 87, 50945098, 1990.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
G - P R O T E I N COUPLED RECEPTORS
533
16.
Schwinn, D.A.; Lomasney,J.W.; Lorenz, W.; Szklut, P.J.; Fremeau, R.T., Jr.; Yang-Fey, T.L.; Caron, M.G.; Lefkowitz, R.J.; and Cotecchia, S. Molecular cloning and expression of the cDNA for a novel al-adrenergic receptor subtype. J. Biol. Chem. 265, 8183-8189, 1990.
17.
Andersen, P.H.; Gingrich, J.A.; Bates, M.D.; Dearry, A.; Falardeau, P.; Senogles, S.E.; and Caron, M.G. Dopamine receptor subtypes: Beyond the D1/D2 receptor classification. Trends in Pharmacol. Sci. (TIPS) 11, 231-236.
18.
Kebabian, J.W.; and Greengard, P. Dopamine-sensitiveadenylate cyclase: A possible role in synaptic transmission. Science 174, 1346-1349, 1971.
19.
Amlaiky, N.; Berger, J.G.; Chang, W.; McQuade, R.J.; and Caron, M.G. Identificationof the ligand binding subunit of the D l dopamine receptor by photoaffinity crosslinking. Mol. Pharmacol. 31, 129-134, 1986.
20.
Cerione, R.A.; Strulovici, B.; Benovic, J.L.; Lefkowitz, R.J.; and Caron, M.G. Pure P-adrenergic receptor: the single polypeptide confers catecholamine responsivenessto adenylate cyclase. Nature 306, 562-566, 1983.
21.
Gingrich, J.A.; Amlaiky, N.; Senogles, S.E.; Chang, W.K.; McQuade, R.D.; Berger, J.G.; and Caron, M.G. Affinity chromatography of the D1 dopamine receptor from rat corpus striatum. Biochemistry 27, 3907-3912, 1988.
22.
Dearry, A.; Gingrich, J.A.; Falardeau, P.; Fremeau, R.T., Jr.; Bates, M.D.; and Caron, M.G. Molecular cloning and expressionof a gene for the human D1 dopamine receptor. Nature in press.
23.
Hausdorff, W.P.; O'Dowd, B.F.; Irons, G.P.; Caron M.G.; and Lefkowitz, R.J. ; Phosphorylation sites on two domains of the P2 adrenergic receptor are involved in distinct pathways of receptor desensitization. J. Biol. Chem. 264, 1265712665, 1989.
24.
Strader, C.D.; Candelore, M.R.; Hill, W.S.; Sigal IS.; and Dixon, R.A.F. Identification of two serine residues involved in agonist activation of the betaadrenergic receptor. J. Biol. Chem. 264, 13572-13578, 1989.
25.
Billard, W.; Ruperto, V.; Crosby, G.; lorio, L.C.; and Barnett, A. Characterization of the binding of [3H]SCH 23390, a selective D1-receptor antagonist ligand, in rat striatum. Life Sci. 35, 1885-1893, 1984.
26.
Spano, P.F.; Tarbucchi, M.; and Di Chiara, G. Localization of nigral doparninesensitive adenylyate cyclase on neurons originating from the corpus striatum. Science 196, 1343-1345, 1977.
27.
Creese, I.; Morrow, L.; Leff, S.; Sibley, D.; and Harnblin, M Dopamine recepors in the central nervous system. Int. Rev. Neurobiol. 23, 255-301, 1982.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 34
GINGRICH ET A L .
28.
Niznik, H. Dopamine receptors: Molecular structure and function. Mol. Cell. Endocrinolgy 54, 1-22, 1987.
29.
Grandy, D.K.; Marchionni,M.A.; Makam, H.; Stofko, R.E.; Atfano, M.; Frothingharn,L.; Fischer, J.B.; Burke-Howie, K.J.; Bunzow, J.R.; Server, A.C.; and Civelli, 0. Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc. Natl. Acad. Sci. USA 86, 9762-9766, 1989.
30.
Baldi, E.; Pupilli, C.; Arnenta, F.; and Mannelli, M. Presence of dopaminedependent adenylate cyclase activity in human renal cortex. Eur. J. Pharrnacol. 149, 351-356, 1988.
3 1.
Missale, C.; Castelletti, L.; Memo, M.; Carruba, M.O.; and Spano, P.F. Identification and characterization of postsynaptic D1 and D2 doparnine receptors in the cardiovascular system. J. Cardiovasc. Pharmocol. 11, 643-650, 1985.
32.
Felder, C.C.; Jose, P.A.; and Axelrod, J. The dopamine agonist, SKF82526, stimulates phospholipase-C activity independent of adenylate cyclase. J. Pharrnacol. Exp. Ther. 248, 171-175, 1989.
JOURNAL OF RECEPTOR RESEARCH, 11(1-4), 521-534 (1991)
MOLECULAR CHARACTERIZATION OF G-PROTEIN COUPLED RECEPTORS: ISOLATION AND CLONING OF A D1 DOPAMINE RECEPTOR
Jay A. Gingrich, Allen Dearry, Pierre Falardeau, Robert T. Fremeau Jr., Michael D. Bates, and Marc G. Caron Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, N.C.
ABSTRACT
This article summarizes the recent progress our laboratory has made in understanding the molecular characteristics of the D1 dopamine receptor. The D1 dopamine receptor from rat striaturn has been purified to near homogeneity using a combination of several chromatographic steps. Furthermore, the gene for the human D1 dopamine receptor has been cloned, sequenced, and expressed. The cloned receptor has all the pharmacologic and biochemical properties of the classical D1 receptor coupled to adenylyl cyclase which has been previously described in the central nervous system.
INTRODUCTION G-protein-coupledreceptors represent a large family of membrane proteins involved in mediating the effects of a large member of hormones and neurotransmitters. Binding of a hormone to one of these receptors promotes the interaction of the ligandreceptor complex with and activation of a heterotrimericG-protein which in turn activates an effector system such as adenylyl cyclase, phospholipase C or an ion channel (1,Z). The biochemical processes by which this signal transduction occurs are fairly well understood. Insights into these processes have been facilitated recently by the elucidation of the primary sequence of the major components of these systems via the isolation and cloning of the genes and cDNA's for these proteins (1 2 ) .
521 Copyright
0 1991 by
Marcel Dekker, Inc.
522
GINGRICH ET A L . The approach initially taken to elucidate the structure of G-protein coupled
receptors such as the adrenergic receptors, was to purify these receptors in sufficient quantities to allow the determinationof partial amino acid sequence (3,4).Based on Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
peptide sequence, oligonucleotide probes were synthesized and cDNA and genes were isolated by recombinant DNA techniques. The first of the receptors to be cloned was the hamster p2 adrenergic receptor (5).Soon after, several other receptors were also cloned such as the pl adrenergic receptor subtype, and several rnuscarinic receptor These proteins all share in common the same seven transrnembrane subtypes (6-9). domain arrangement (4).Within these transrnembrane domains, these proteins display considerable amino acid identity, varying from almost 75% within subtypes such as pl and p2 adrenergic receptors to 30-35% when comparing distant receptors such as p adrenergic and muscarinic receptors (10). This extensive sequence conservation has been used as a strategy in the cloning of several related members of this family of proteins (1,11,12). The cDNAs and/or genes for a large number of G-protein coupled receptors have been isolated thus far. Whereas all of these receptors appear to be very similar in their structural properties, an unexpected finding has been the large number of previously unsuspected receptor subtypes revealed by the molecular biology approach.
For example, previously, only four different adrenergic receptors had been distinguished by pharmacologicaland biochemical studies. It is now evident from the isolation of cDNAs and genes, that at least 9 adrenergic receptors exist, three p subtypes, three a1 subtypes, and three a2 subtypes (1,13-16).The exact physiologicalrole that these various receptor subtypes play remains to be examined. DOPAMINE RECEPTORS. In the 25 years since dopamine was identified as a
neurotransmitterin the CNS, great progress has been made towards understandingthe role of dopaminergic systems in normal and pathologic states. The effects of dopamine are presumably mediated by two distinct G-protein coupled receptors: D1 receptors that are coupled to stimulation of adenylyl cyclase and D2 receptors that inhibit the enzyme but also activate K+ channels (17). However, in contrast to the wealth of studies describing the functional effects of dopamine, insights into the biochemistry of the receptors which mediate these effects have lagged far behind. This generalization is especially true for the D1 dopamine receptor. Although the first dopamine-stimulated activation of adenylyl cyclase was described in 1971 (18), very little progress has been
G-PROTEIN COUPLED RECEPTORS
523
made towards understanding the biochemistry of this receptor. Beginning 7 or 8 years ago our group initiated an approach to elucidate the molecular events underlying the effects of dopamine acting through D1 and D2 dopamine receptors. Our specific Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
interest was in understanding the molecular properties of these receptor proteins, and in this brief report, we review some of the progress towards this goal which our group has made with the D1 receptor subtype.
RESULTS IDENTIFICATION AND PURIFICATION OF THE D7 DOPAMINE RECEPTOR.
In
collaboration with scientists at the Schering-PloughCorporation, we sought to develop a D1 selective benzazepine antagonist which could be used as a photoaffinity probe for this receptor. The result of this collaboration was the compound, [1251]SCH39582, which could be photo-crosslinked to the D1 receptor by the bifunctional reagent, SANPAH (19). Using this technique, the D1 receptor was identified to be a 72 kDa glycoprotein (see Figure 1A) and from work with the 82 adrenergic receptor (20), it was expected that the ligand binding subunit identified by this photoprobe would also possess the adenylyl cyclase coupling capacity of this receptor as well. One of the most powerful tools in biochemistry has proven to be the ability to study the properties of a pure protein in in vitro assays. For this reason we undertook the purification of the D1 receptor. With the development of a D1 selective antagonists and which could photo-crosslink the D1 receptor, we believed that these compounds could be adapted for use as biospecific affinity chromatography ligands. For this purpose, SCH39111 was synthesized. When immobilized to Sepharose beads through an appropriate spacer arm, SCH39111 was able to function as a biospecific affinity ligand capable of 200-300 fold purification of the receptor from crude digitonin-solubilizedrat striatal membranes (21). Further purification (Figure 1B) of this partially purified receptor using ion exchange, size exclusion, and lection chromatographyyielded a preparation with a specific activity of ca. 11,000 pmol [3H]SCH23390boundhng protein (theoretical specific activity being approximately 13,000 pmollmg). When this preparation was radioiodinatedusing Bolton-Hunterreagent, a predominant band at 72 kDa was observed after SDS-PAGE and autoradiography (Figure 1C). Thus, using this
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 24
GINGRICH ET A L .
FIGURE 1. Identification and purification of the rat striatal D1 dopamine receptor. A. A n autoradiogram of an SDS-PAGE of D1 receptor crosslinkedto [1251)SCH39582 using the bifunctional reagent. SANPAH. In the absence (-) and presence (+) of 1 fl (+) butachfml. The radoligand is imrporated into a protein wdh an apparent mass of 72 kDa B. An Outline of the procedure used to obtain highly puriiied D1 receptor. C. An autoradiogram of an SDS-PAGE aiter treating the purified receptor preparation(specific activily = ca. 11,000 pmol [3H]SCH23390binding siteslrng protein) wth [1251]Bolton-Hunterreagent. Note that the predominant protein in this preparationis the 72 kDa band which wmgrates with photoatfinity labelled receptor.
procedure, we were successful in obtaining highly purified preparations of the D1 receptor. CLONING
OF THE GENE FOR THE D1 DOPAMINE RECEPTOR. As summarized in
the introduction, the application of molecular biology to receptor research has proven extremely fruitful in the last several years. An early hope during the course of these studies was that purification might provide enough receptor protein to allow partial peptide sequence analysis of this receptor. However, given the difficulty in obtaining
G - P R O T E I N COUPLED RECEPTORS
525
the large amounts of pure receptor protein necessary for this purpose, an alternate strategy was employed to isolate the gene and cDNA for the D1 receptor. Taking advantage of the sequence information recently described for the D2 dopamine Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
receptor (12), we designed an oligonucleotide probe based on its putative second transmembrane domain and used this probe to screen a human retina cDNA library. The second transmembrane domain was chosen because it is highly conserved between other receptor subtypes and was thus thought likely to be conserved between the D2 and D1 receptors. A retinal library was chosen because dopamine is the predominant catecholaminein this tissue. This screening yielded several D2 clones as well a unique clone (D233) which upon sequencing, appeared to encode a novel member of the Gprotein coupled receptor family. The clone D233 was not full length, apparently lacking the amino terminus and the first transmembrane domain. A full length clone (HGL26) was obtained after screening a human genomic library with a 700 bp Pvu II fragment of D233. Sequencing of HGL26 confirmed its identity with D233, and also demonstrated that the gene for this novel receptor was intronless within its coding region (a result predicted from genomic Southern analysis). The deduced amino acid sequence and the predicted transmembrane topology of the open reading frame of HGL26 are shown in Figure 2 (22). Several features of this receptor sequence are of note. First, this receptor fits the well established pattern of seven hydrophobic segments (thought to be membrane spanning) which are found in all known G-protein coupled receptors. The protein also possesses 2 consensus sequences for N-linked glycosylation on its proposed extracellular domains. Furthermore, several consensus sites for protein kinases A and C as well as potential sites for agonist dependent receptor kinases such as the p adrenergic receptor kinase (23) are found in the proposed intracellular domains. That this receptor might be a catecholamine receptor seemed likely since this protein possessed the aspartate and two serine residues thought to be involved in catecholamine binding (24). However, the identification of this gene as the human D1 receptor would depend on analysis of the expressed protein. EXPRESSION OF THE CLONED D1 RECEPTOR GENE. To study the properties of
the protein encoded by HGL26, the coding region was subcloned into the mammalian expression vector pCMV5 in both a sense (pCMV5IDl) and antisense (pCMV5/Dl') orientation and used to transfect monkey kidney cells (COS7) cells in a transient fashion.
transmembrane domains are those traversing the stippled gray bar (plasma membrane). Potential sites of N-linked glycosylation are indicated by the "Y"-shaped structures made of small circles. Consensus sites for CAMP dependent protein kinase (A) and protein kinase C (C) phosphorylation are shown by the darkened circles.
FIGURE 2. Primary sequence and putative transmembrane topolgy of the human D1 dopamine receptor. The primary sequence is represented by single letter codes for each amino acid. The predicted
In tracellular
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
U
r
d
n
2
n
n
G-PROTEIN COUPLED RECEPTORS
527
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
TABLE I Antaaonists;
!wm!!a
SCH23390 cis pifluthixol (+) butaclamol cis flupenthixol SCH23388 ketanserin spiperone prazosin (-) butaclamol alprenoloI raclopride
0.1 1 0.20 0.90 1.6 22 190 220 3,000 4,900 16,000 >72,000
Aaonlsts: 20 fenoldopam SK F38393 87 210 (-)apomorphine 2,500 dopamine quinpirole 14,000 serotonin >15,000 epinephrine >55,000
Ligand Binding Pharmacology of the Cloned D1 Dopamine Receptor Expressed In COS-7 Cells. Cos-7 cells were transfected as described (22). Membranes prepared from these cells display saturable high affinity binding sites for [1251]SCH23982(0.35 nM, n=6). Various compounds were tested for their potency in competition assays with [1251]SCH23982. The results are expressed here as the average of at least three determinations. Agonists did not display biphasic curves in crude membrane preparations.
Figure 3 shows the result obtained when several Vitiated ligands specific for various types of biogenic amine receptors were tested on membranes from transfected cells. As shown, [3H]SCH23390 binding sites were increased ca. 20-fold on cells transfected with the sense construct compared with the antisense control. The binding sites for other ligands were not significantly increased, suggesting that HGL26 might be encoding the D1 dopamine receptor. Supporting this hypothesis, the binding of I3H]SCH23390 and its related compound [1251]SCH23982was found to be saturable and of high affinity (0.10 and 0.35 nM respectively). Furthermore,when several compounds were tested
for their ability to compete for [1251]SCH23982 binding to the expressed receptor, the order of potency observed in these experiments was entirely consistent with the pharmacology of the D l receptor from the caudate and putamen (25). The observed dissociation constants (Kds) for several of these compounds are summarized in Table I. Thus on the basis of ligand binding properties alone, the identity of this cloned receptor can be safely secured as the D1 dopamine receptor.
GINGRICH ET A L .
525
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
6000
5000
'
E 3000 Q.E 0-
1000
C
SCH SPIRO KET
YOH
ALP
FIGURE 3. Identificationof HGL26 protein product as the D1 dopamine receptor using ligand binding. Cells (cos-7) were transfected transiently with either the sense (pCMWD1) or antisense (pCMWD1') constructs and membrane homogenates were tested for tritiated ligand (1 nM +/- 10 pM competitor) binding 48 hr later. As shown cells transfected with the sense construct bound 18-fold more tritiated SCH23390 than cells transfected with the antisense construct . In contrast, the binding of other tritiated antagoniststested was similar in each transfection condition. SCH; SCH 23390 competed with cis flupenthixol, SPIRO; spiroperidol competed with cis flupenthixol, KET; ketanserin competed with serotonin, YOH; yohimbine competed with epinephrine, ALP; alprenolol competed with epinephrine.
G-PROTEIN COUPLED RECEPTORS
529
TABLE II
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
Conditions: basal dopamine dopamine+SCH23390 doparnine+raclopride dopamine+propranolol SKF38393 quinpirole
adenylyl cyclase actlvlty fold stlmulatlon (pmollmg protein 60 min) 235*5 1079k55 3 19+24 1128f129 1049k43 605k49 285+8
1 .o 4.6 1.4 4.8 4.5 2.6 1.2
Signal Transductlon of the Cloned D1 Receptor Expressed in Mouse L(TK) cells. L(Tk-) cells were cotransfectedwith pCMV5ID1 and a neomycin resistance plasmid. Clonal cells were selected which possessed G418 resistance. Membranes from these cells were assayed for dopamine stimulated adenylyl cyclase activity by previously described methods using maximally effective doses of agonist dopamine = 100 FM ; all other compounds were present at 1 bM). Notice that KF38393 is only a partial agonist at the D1 dopamine receptor for adenylyl cyclase stimulation which is in accord with previous results with this compound.
L
To assess the second messenger system of this cloned D1 dopamine receptor,
pCMVUD1 was used to permanently drive the expression of the D1 receptor in mouse L(tk-)cells, a host normally devoid of this receptor. Clonal cell lines were isolated which expressed 50-500 fmol receptorhng protein and these cells were then tested for their ability to stimulate the adenylyl cyclase second messenger system. Table II demonstrates that cells expressing this receptor are able to stimulate adenylyl cyclase activity 5-fold over basal and that this activity has all the pharmacologic properties expected of the D1 dopamine receptor. Importantly, no dopamine stimulated adenylyl cyclase activity can be elicited in untransfectedcells.
LOCALIZATION OF THE MESSAGE FOR THE DI DOPAMIME RECEPTOR.
Several techniques such as northern blot analysis, in situ hybridization, and polymerase chain reaction (PCR) were used to detect the messenger RNA for the D1 receptor in various peripheral organs and brain regions. All of these methods in concert yield a consistent picture of receptor expression. Message for this recept has the highest expression in the caudate nucleus, nucleus accumbens, and olfactory tubercle-- with
GINGRICH ET A L .
5 30
frontal cortex and hippocampus showing somewhat lower levels. Interestingly, two tissues which have relatively high levels of D1 dopamine receptors have undetectable levels of receptor message: substantia nigra and kidney. In the case of the substantia Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
nigra, this finding is consistent with the previously held hypothesis developed from lesioning studies (26) that D l dopamine receptors in this tissue exist on presynaptic nerve terminals whose cell bodies originate in the caudate. In the other instance, the kidney is known to possess postsynaptic D1 dopamine receptors, and thus the failure to find receptor message in this tissue (using the sensitive technique of PCR amplification as well as traditional northern blot analysis) suggests that this tissue may well contain a distinct but closely related subtype of the D1 receptor. This hypothesis is consistent with numerous pharmacologic discrepancies which have been noted between central and peripheral D1 dopamine receptors
DISCUSSION In summary, over the last several years our laboratory has progressedfrom our early attempts to learn more about the molecular characteristicsof the D1 receptor through photoaffinity labelling and purification of this receptor to elucidation of its deduced primary sequence and gene structure using molecular biological techniques. The tools are now in place to begin the process of studying the molecular aspects of signal transduction and regulation of this receptor. As summarized in this paper the human D1 dopamine receptor characterized here has all the properties of the classical D1 dopamine receptor characterized in the striaturn of several species (27,28). This receptor is typical of other G-protein coupled receptors. Overall, the D1 dopamine receptor sequence is most similar to the p l adrenergic receptor closely followed by the 02 dopamine and p2 adrenergic receptors. This emphasizes the fact that although D1 and D2 receptors both bind dopamine, they are quite different at the biochemical level. This is further highlighted by the fact that the D1 receptor appears to be intronless within its coding region, whereas the 0 2 receptor has several introns (29). One of the more interesting aspects of this work is the evidence which points to the possible presence of other subtypes of D1 dopamine receptors. The first evidence as mentioned above is the incongruence between message and receptor binding sites as observed in the kidney. A second line of evidence comes from the signal
G-PROTEIN COUPLED RECEPTORS
531
transduction studies. In mouse L(tk-) cells expressing the D i receptor, dopamine promotes stimulation of adenylyl cyclase or accumulation of intracellular CAMP but does not elicit stimulation of phosphatidylinositol hydrolysis (22). However, there is evidence Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
both from studies with striatal and kidney preparations (30-32) that dopamine through a D1 receptor can stimulate accumulation of diacylglycerol and inositol trisphosphate
under certain conditions. This suggests that a D1 receptor distinct from cloned brain receptor may exist that is coupled selectively to the phosphatidylinositol hydrolysis pathway. Thus, even at this early stage of characterizationof this receptor, evidence suggests that this receptor may be part of a larger family of D1 dopamine receptors. These findings are consistent with several other pharmacological, biochemical, electrophysiological and behavioral considerations (17). The tools are now in place to attempt to shed light on this heterogeneity and to study the molecular aspects of signal transduction through this receptor as well as the regulation of its responsiveness that may be a key element of its role in certain disorders.
ACKNOWLEDGEMENTS We thank V. Clack and N. Godinot for technical assistance. This work was supported in part by NIH grant NS19576. J.A.G. and M.D.B. are both fellows in the Medical Scientist Training Program.
REFERENCES 1.
Lefkowitz, R.J.; and Caron, M.G. Adrenergic receptors: Models for the study of receptors coupled to guanine nucleotide regulatory proteins. J. Biol. Chem. 263, 4993-4996, 1988.
2.
Casey, P.J.; and Gilman, A.G. G-protein involvement in receptor-effector coupling . Ann. Rev. Biochem. 56, 2577-2581, 1988.
3.
Benovic, J.L.; Shorr, R.G.L.; Caron, M.G.; and Lefkowitz, R.J. The mammalian p2 adrenergic receptor: purification and characterization. Biochemistry 23, 4510-4518, 1984.
4.
Dohlman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry 26, 2657-2664, 1987.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 32
GINGRICH ET A L .
5.
Dixon, R.A.F.; Kobilka, B.K.; Strader, D.J. ; Benovic, J.L.; Dohlman, H.G.; Frielle, T.; Bolanowski, M.A.; Bennett, C.D.; Rands, E.; Diehl, R.E.; Mumford, R.A.; Slater, E.E.; Sigal, IS.; Caron, M.G.; Lefkowitz, R.J.; and Strader, C.D. Cloning of the gene and cDNA for mammalian P-adrenergic receptor and homology to rhodopsin. Nature 321, 75-79, 1986.
6.
Yarden, Y.; Rodriguez, H.; Wong, S.K.-F.; Brandt, D.R.; May, D.C.; Bumier, J.; Harkins, R.N.; Chen, E.Y.; Ramachandran, J.; Ullrich, A,: and Ross, E.M. The avian P-adrenergic receptor: Primary structure and membrane topology. Proc. Natl. Acad. Sci. USA 83, 6795-6799, 1986.
7.
Kub,T.; Fukuda, K.; Mikami, A,; Maeda, A.; Takahashi, H.; Mishina, M.; Haga, T.; Haga, k.; Ichiyama, A.; Kangawa, K.; Kojima, M.; Matsuo, H.; Hirose, T.; and Numa, S. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323, 411-416, 1986.
8.
Kubo,T.; Maeda, A.; Sugimoto, K.; Akiba, 1.; Mikami, A.; Takahashi, H.; Haga, T.; Haga, K.; Ichiyama, A.; Kangawa, K.; Matsuo, H.; Hirose, T.; and Numa, S. Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence. FEBS. Lett. 209, 367-373, 1986.
9.
Bonner, T.I.; Buckley, N.J.; Young, A.C.; and Brann, M.R. Identificationof a family of muscarinic acetylcholine receptor genes. Science 237, 527-532, 1987.
10.
ODowd, B.F.; Caron, M.G.; and Lefkowitz, R.J. Structure of the adrenergic and related receptors. Ann. Rev. Neurosci. 12, 67-83, 1989.
11.
Kobilka, B.K.; Frielle, T.; Collins, S.; Yang-Feng, T.; Kobilka, T.S.; Franke, U.; Lefkowitz, R.J.; and Caron, M.G. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329, 75-79, 1987.
12.
Bunzow, J.R.; VanTol, H.H.M.; Grandy, D.K.; Albert, P.; Salon, J.; Christie, M.; Machida, C.A.; Neve, K.A.; and Civelli, 0. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature, 336, 783-787, 1988.
13.
Emorine, L.J.; Marullo, S . ; Briend-Sutren,M.M.; Patey, G.; Tate, K.; DelavierKlutchko, C.; and Strosberg, A.D. Molecular characterizationof the human beta 3-adrenergic receptor. Science 245, 1118-1121, 1989.
14.
Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; and Kobilka, B.K. Cloning and expressionof a human kidney cDNA for an a2 adrenergic receptor subtype. Proc. Natl. Acad. Sci USA 85, 6301-6305, 1988.
15.
Lomasney, J.W.; Lorenz, W.; Allen, L.F.; King, K.; Regan, J.W.; Yang-Feng, T.L.; Caron, M.G.; and Lefkowitz, R.J. Expansion of the a2-adrenergic receptor family: Cloning and characterizationof a human a2 receptor subtype, the gene for which is located on chromosome 2. Proc. Natl. Acad. Sci. USA 87, 50945098, 1990.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
G - P R O T E I N COUPLED RECEPTORS
533
16.
Schwinn, D.A.; Lomasney,J.W.; Lorenz, W.; Szklut, P.J.; Fremeau, R.T., Jr.; Yang-Fey, T.L.; Caron, M.G.; Lefkowitz, R.J.; and Cotecchia, S. Molecular cloning and expression of the cDNA for a novel al-adrenergic receptor subtype. J. Biol. Chem. 265, 8183-8189, 1990.
17.
Andersen, P.H.; Gingrich, J.A.; Bates, M.D.; Dearry, A.; Falardeau, P.; Senogles, S.E.; and Caron, M.G. Dopamine receptor subtypes: Beyond the D1/D2 receptor classification. Trends in Pharmacol. Sci. (TIPS) 11, 231-236.
18.
Kebabian, J.W.; and Greengard, P. Dopamine-sensitiveadenylate cyclase: A possible role in synaptic transmission. Science 174, 1346-1349, 1971.
19.
Amlaiky, N.; Berger, J.G.; Chang, W.; McQuade, R.J.; and Caron, M.G. Identificationof the ligand binding subunit of the D l dopamine receptor by photoaffinity crosslinking. Mol. Pharmacol. 31, 129-134, 1986.
20.
Cerione, R.A.; Strulovici, B.; Benovic, J.L.; Lefkowitz, R.J.; and Caron, M.G. Pure P-adrenergic receptor: the single polypeptide confers catecholamine responsivenessto adenylate cyclase. Nature 306, 562-566, 1983.
21.
Gingrich, J.A.; Amlaiky, N.; Senogles, S.E.; Chang, W.K.; McQuade, R.D.; Berger, J.G.; and Caron, M.G. Affinity chromatography of the D1 dopamine receptor from rat corpus striatum. Biochemistry 27, 3907-3912, 1988.
22.
Dearry, A.; Gingrich, J.A.; Falardeau, P.; Fremeau, R.T., Jr.; Bates, M.D.; and Caron, M.G. Molecular cloning and expressionof a gene for the human D1 dopamine receptor. Nature in press.
23.
Hausdorff, W.P.; O'Dowd, B.F.; Irons, G.P.; Caron M.G.; and Lefkowitz, R.J. ; Phosphorylation sites on two domains of the P2 adrenergic receptor are involved in distinct pathways of receptor desensitization. J. Biol. Chem. 264, 1265712665, 1989.
24.
Strader, C.D.; Candelore, M.R.; Hill, W.S.; Sigal IS.; and Dixon, R.A.F. Identification of two serine residues involved in agonist activation of the betaadrenergic receptor. J. Biol. Chem. 264, 13572-13578, 1989.
25.
Billard, W.; Ruperto, V.; Crosby, G.; lorio, L.C.; and Barnett, A. Characterization of the binding of [3H]SCH 23390, a selective D1-receptor antagonist ligand, in rat striatum. Life Sci. 35, 1885-1893, 1984.
26.
Spano, P.F.; Tarbucchi, M.; and Di Chiara, G. Localization of nigral doparninesensitive adenylyate cyclase on neurons originating from the corpus striatum. Science 196, 1343-1345, 1977.
27.
Creese, I.; Morrow, L.; Leff, S.; Sibley, D.; and Harnblin, M Dopamine recepors in the central nervous system. Int. Rev. Neurobiol. 23, 255-301, 1982.
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
5 34
GINGRICH ET A L .
28.
Niznik, H. Dopamine receptors: Molecular structure and function. Mol. Cell. Endocrinolgy 54, 1-22, 1987.
29.
Grandy, D.K.; Marchionni,M.A.; Makam, H.; Stofko, R.E.; Atfano, M.; Frothingharn,L.; Fischer, J.B.; Burke-Howie, K.J.; Bunzow, J.R.; Server, A.C.; and Civelli, 0. Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc. Natl. Acad. Sci. USA 86, 9762-9766, 1989.
30.
Baldi, E.; Pupilli, C.; Arnenta, F.; and Mannelli, M. Presence of dopaminedependent adenylate cyclase activity in human renal cortex. Eur. J. Pharrnacol. 149, 351-356, 1988.
3 1.
Missale, C.; Castelletti, L.; Memo, M.; Carruba, M.O.; and Spano, P.F. Identification and characterization of postsynaptic D1 and D2 doparnine receptors in the cardiovascular system. J. Cardiovasc. Pharmocol. 11, 643-650, 1985.
32.
Felder, C.C.; Jose, P.A.; and Axelrod, J. The dopamine agonist, SKF82526, stimulates phospholipase-C activity independent of adenylate cyclase. J. Pharrnacol. Exp. Ther. 248, 171-175, 1989.
