Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the Gq pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain. Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the Gq pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain. The dopamine D1 receptor has been shown to signal via Gs and Golf proteins to stimulate adenylyl cyclase (1Zhuang X. Belluscio L. Hen R. J. Neurosci. 2000; 20: 1-5PubMed Google Scholar), and there have been reports of calcium signaling by D1 receptor-mediated phosphatidylinositol hydrolysis by phospholipase C (PLC), 1The abbreviations used are: PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; PTX, pertussis toxin. presumably through Gq coupling. Although PLC activity was not detected in studies on the D1 receptor expressed in COS7 cells (2Dearry A. Gingrich J.A. Falardeau P. Fremeau Jr., R.T. Bates M.D. Caron M.G. Nature. 1990; 347: 72-76Crossref PubMed Scopus (593) Google Scholar) or in Chinese hamster ovary and baby hamster kidney cells (3Pedersen U.B. Norby B. Jensen A.A. Schiodt M. Hansen A. Suhr-Jessen P. Scheideler M. Thastrup O. Andersen P.H. Eur. J. Pharmacol. 1994; 267: 85-93Crossref PubMed Scopus (33) Google Scholar), it has been shown that activation of the D1 receptor in brain tissue (4Undie A.S. Friedman E. J. Pharmacol. Exp. Ther. 1990; 253: 987-992PubMed Google Scholar, 5Pacheco M. Jope R. J. Neurochem. 1997; 69: 639-644Crossref PubMed Scopus (47) Google Scholar) or in Xenopus oocytes injected with mRNA taken from striatum (6Mahan L.C. Burch R.M. Monsma Jr., F.J. Sibley D.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2196-2200Crossref PubMed Scopus (179) Google Scholar) resulted in phosphatidylinositol turnover. This discrepancy suggests the involvement of a factor in D1 receptor-mediated PLC activity that is present in native tissues but absent in heterologous expression systems. An examination of the above studies where PLC activation was observed indicates that, although D1 receptor-selective antagonists blocked phosphatidylinositol turnover demonstrating the involvement of the D1 receptor, the amount of agonist used to elicit the response was greater than 100 μm, a concentration at which even selective ligands may bind other receptors. For example, SKF 81297, a highly D1-selective agonist, has a Ki for the D1 receptor of ∼1 nm but has a Ki for the D2 receptor of ∼900 nm (7Neumeyer J.L. Kula N.S. Bergman J. Baldessarini R.J. Eur. J. Pharmacol. 2003; 474: 137-140Crossref PubMed Scopus (84) Google Scholar). This analysis suggests the possibility that the D1 agonist-mediated PLC stimulation by high concentrations of drugs observed in physiological systems may have resulted from the coincident activation of another receptor. There are considerable data indicating that the dopamine D1 and D2 receptors are functionally linked. For example, behavioral studies have shown that co-stimulation of the D1 receptor is essential for D2 agonists to produce maximal locomotor stimulation (8Dreher J.K. Jackson D.M. Brain Res. 1989; 487: 267-277Crossref PubMed Scopus (166) Google Scholar) and that activation of both D1 and D2 receptors was required to augment the acute effects of cocaine action (9Kita K. Shiratani T. Takenouchi K. Fukuzako H. Takigawa M. Eur. Neuropsychopharmacol. 1999; 9: 1-7Crossref PubMed Scopus (41) Google Scholar). Biochemical and electrophysiological evidence has also supported D1-D2 receptor synergism. Combined administration of specific D1 and D2 agonists potentiated immediate early gene expression (10Svenningsson P. Fredholm B.B. Bloch B. Le Moine C. Neuroscience. 2000; 98: 749-757Crossref PubMed Scopus (38) Google Scholar, 11Gerfen C.R. Keefe K.A. Gauda E.B. J. Neurosci. 1995; 15: 8167-8176Crossref PubMed Google Scholar). Also, long term depression of synaptic transmission after dopamine depletion could be restored by dopamine or co-administration of specific D1 and D2 agonists but not by either selective agonist alone (12Calabresi P. Maj R. Mercuri N.B. Bernardi G. Neurosci. Lett. 1992; 142: 95-99Crossref PubMed Scopus (169) Google Scholar). Although the mechanism for the D1-D2 receptor interaction in these studies showing functional linkage is not clearly understood, there is evidence that an interaction between the receptors occurring within the same cell may be involved in some instances. For example, synergistic potentiation of the D2 receptor-mediated enhancement of arachidonic acid release by co-administration of D1-selective and D2-selective agonists in cells expressing both receptors has been observed (13Piomelli D. Pilon C. Giros B. Sokoloff P. Martres M.P. Schwartz J.C. Nature. 1991; 353: 164-167Crossref PubMed Scopus (245) Google Scholar). Furthermore, it has been shown that D1 subclass and D2 subclass receptors are co-expressed in neurons of the rat striatum (14Aizman O. Brismar H. Uhlen P. Zettergren E. Levey A.I. Forssberg H. Greengard P. Aperia A. Nat. Neurosci. 2000; 3: 226-230Crossref PubMed Scopus (335) Google Scholar), suggesting the possibility that there may be a direct D1-D2 receptor association. Therefore, we postulated that dopaminergic PLC-mediated calcium signaling results from the co-activation of dopamine D1 and D2 receptors and the formation of a novel signaling unit represents the molecular basis for the functional interaction between these receptors. In investigating this hypothesis, we demonstrated that co-activation of co-expressed D1 and D2 receptors resulted in a PLC-mediated increase of intracellular calcium levels, a signaling pathway not activated when either one of the receptors was singly activated. Further, we also found evidence that D1 and D2 receptors associate within neurons and thereby potentially form a novel signaling complex. Cell Culture and Receptor Expression—All cell culture and transfection reagents were obtained from Invitrogen. COS7 and HEK293T cells were maintained as monolayer cultures at 37 °C in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. Transient expression was performed using LipofectAMINE™ with DNA encoding human D1 receptor or the long isoform of the human dopamine D2 receptor that had been inserted into pcDNA3 vector (Invitrogen). For immunoprecipitation experiments, HA and FLAG epitope tags were introduced after the initiation methionine of the D1 and D2 receptors, respectively, by PCR. Stable cell lines co-expressing the N terminus HA epitope-tagged D1 receptor and N terminus FLAG epitope-tagged human D2 receptor were created in HEK293TSA cells utilizing the pBudCE 4.1 vector (Invitrogen). Briefly, the D1 receptor cDNA was inserted into the EF1α multicloning site and the D2 receptor cDNA into the cytomegalovirus site. Antibiotic-resistant clones (selected with 200 μg/ml zeocin) of each transfection were isolated and tested for expression of corresponding receptors using saturation binding analysis. Measurement of Calcium Signal—Calcium mobilization assays were carried out using a FLEXstation multiwell plate fluorometer (Molecular Devices, Sunnyvale, CA). Stably transfected cells were seeded in black microtiter plates at a density of ∼105 cells/well and grown for 24 h. The cells were then with (Molecular in medium supplemented with and for and with and (Invitrogen). were for and in corresponding to in intracellular calcium levels the of agonists were also were at for For of the for each agonist concentration were and using CA). In signaling pathway cells were with or nm for or with μg/ml pertussis toxin for to the calcium cyclase assays were as C. M. PubMed Scopus Google Scholar). The of of of unit of and of in a of For the experiments, was in the The was with to SKF or for and were were by of cultures from the striatum were using Briefly, tissue was from and with and The tissue was then in a of and for at 37 by in with were by in a and and then This was by in medium minimal essential medium with and 10% fetal bovine were on and in plates at a density of ∼105 The following the medium was with A medium supplemented with and (Invitrogen). the medium was with medium analysis of neurons was performed after in of Brain brain tissue was obtained from the Brain and for at the of of Brain tissue from was also the Neuroscience. 1994; PubMed Scopus Google Scholar). The for the D1 receptor was obtained from and the receptor was from to D1 and D2 receptors were with and tetramethylrhodamine (Molecular was performed using a was tested using of cells expressing each of the dopamine receptor was tested by in which was of were in in for at 37 in and then blocked for at in 10% bovine serum in were to each in a of the and cells were at The cells were and with for at another of the were on The used in the were receptor and receptor of Receptor vector encoding epitope-tagged human dopamine receptor or encoding HA epitope-tagged human dopamine receptor were via and into pcDNA3 A FLAG epitope-tagged human dopamine receptor cDNA was by of and the for and cells were singly transfected with one of these or with an expression vector encoding either the D1 receptor or the D2 receptor The cells were on in a to the one above for The receptors expressed in these cells were then by using the D1 or D2 receptor from and respectively, and using an corresponding to the epitope of the receptor. from HEK293T cells was performed as T. R. G. J. 2000; PubMed Scopus Google Scholar), the and were used for of the HA and For immunoprecipitation from tissue was from and the was carried out using a M. J. J. 2003; PubMed Scopus Google Scholar). were and with of D1 or D2 protein was by and then for analysis. were using a D1 receptor of D1 and D2 the co-activation of dopamine D1 and D2 receptors resulted in a calcium cells co-expressing D1 and D2 receptors were by administration of concentrations of SKF 81297, a D1-selective agonist, and the D2-selective agonist a significant increase in intracellular calcium levels was observed A and Calcium mobilization was detected following agonist and the signal within of the to agonist This maximal response was a nm concentration of each of the selective agonists to of the maximal response of the cells co-expressing the D1 and D2 receptors with either SKF or not increase intracellular calcium levels significant in intracellular calcium levels could be observed in cells expressing only the D1 receptor that were with SKF or or with both agonists there was calcium signal by SKF 81297, or the co-administration of both drugs in cells expressing only the D2 receptor For of the of the calcium activation of receptors was with that of the D1 and D2 receptors. cells have been shown to and receptors that stimulate PLC via Gq coupling when activated 1997; PubMed Scopus Google Scholar). the of calcium signal is distinct for these two receptors, the receptor-mediated signal at a than the signal by receptors J. Neurosci. 2003; PubMed Google Scholar). The increase in intracellular by the D1 and D2 receptor co-activation was to that detected for receptors activated by the for activation of both D1 and D2 receptors in to calcium signal in cells expressing both D1 and D2 receptors, cells co-expressing D1 and D2 receptors were with either a D1-selective or with a D2-selective the calcium signal was by the of or by the of We that dopamine treatment elicit calcium increase in a to that by administration of SKF and in intracellular calcium levels was by dopamine in cells expressing only D1 or D2 dopamine when cells co-expressing D1 and D2 receptors were with an increase in intracellular calcium levels was the intracellular calcium increase could be blocked by the of or In to coupling to the adenylyl cyclase signaling pathway was by of D1 and D2 receptors, we cells co-expressing D1 and D2 receptors with SKF or with The of the D1 receptor to stimulate adenylyl cyclase and of the D2 receptor to adenylyl cyclase was There was significant in the stimulation of adenylyl cyclase and the between the D1-D2 cells and cells expressing only the D1 receptor. The same was for the of adenylyl and the between the D1-D2 cells and cells expressing only the D2 receptor. that cells expressing both receptors may be of distinct for D1-D2 Receptor to the mechanism of the calcium signal we to of several signal the receptors with both and D2-selective Gs protein activation by the D1 receptor has been in calcium signaling via protein kinase A M. Pharmacol. 1995; Google Scholar, O. 1992; PubMed Scopus (41) Google Scholar, J. Jr., Greengard P. 1995; PubMed Scopus Google Scholar). Therefore, cells expressing D1 and D2 receptors were with an inhibitor of PKA, and co-activated by D1 and D2 receptor There was of on the increase in intracellular calcium levels, suggesting that co-activated D1 and D2 receptor-mediated calcium signaling was not on We then activated D1 and D2 receptors increase intracellular calcium levels via a pathway. were with a PLC the increase in intracellular calcium levels by D1 and D2 receptor co-activation was after treatment with also the calcium signal by the receptors indicating that the inhibitor was results that, when D1 and D2 receptors may to Gq and signal through an pathway to the release of intracellular receptor stimulation of PLC may also via a protein kinase C mechanism J. PubMed Scopus Google Scholar). Furthermore, there is evidence that of action may a PLC-mediated calcium signal K. A. L.C. J. PubMed Scopus Google Scholar). We or a in the calcium signal by co-activated D1 and D2 receptors. cells co-expressing D1 and D2 receptors were with a and with D1 and D2 of calcium signal was observed suggesting that PLC was not by the of when we the effects of the on the D1-D2 receptor-mediated calcium significant were suggesting for in the novel signal by D1-D2 receptor Although the the proteins with D2 receptor signal have not been in of intracellular calcium levels, the pertussis toxin signaling have been to the activation of calcium In of proteins have been shown to stimulate PLC in PubMed Scopus Google Scholar) or by the Gq which in PLC K. Pharmacol. 2003; PubMed Scopus Google Scholar). We the co-activation of D1 and D2 receptors resulted in calcium signaling via a pathway. of cells co-expressing the D1 and D2 receptors with resulted in a in the calcium stimulation by co-administration of SKF and A and This not to be on the concentration of PTX, the of was for of we have shown that μg/ml adenylyl cyclase B. and S. R. but concentrations of to μg/ml not D1-D2 receptor-mediated calcium In receptors, receptor-mediated calcium signal in cells was also blocked by that may the calcium signal by co-activated D1-D2 receptors in a but that the pathway is not involved in the of the of D1 and D2 to the of D1 and D2 receptors resulted in a in the of the binding of either receptor, binding studies were of selective binding by dopamine or selective agonists significant in binding when D1 and D2 dopamine receptors were co-expressed not In saturation binding there was in the of the antagonists or for the D1 and D2 receptors, respectively, The and for binding were protein and nm for the cells expressing the D1 receptor alone and were protein and nm for cells expressing both D1 and D2 receptors. The and for binding were protein and nm for D2 cells and and nm for cells expressing D1 and D2 receptors. results that not the binding of the D1 or the D2 receptors. D1 and D2 in to there was a physiological basis for the novel response by D1 and D2 receptor we these two receptors were co-expressed and co-localized within the same The expression of D1 and D2 receptors within neurons in brain tissues and neurons was and receptors have been shown to be co-localized in neurons of the striatum (14Aizman O. Brismar H. Uhlen P. Zettergren E. Levey A.I. Forssberg H. Greengard P. Aperia A. Nat. Neurosci. 2000; 3: 226-230Crossref PubMed Scopus (335) Google there has been some as to the D1 and D2 receptor are co-expressed in the same cell (14Aizman O. Brismar H. Uhlen P. Zettergren E. Levey A.I. Forssberg H. Greengard P. Aperia A. Nat. Neurosci. 2000; 3: 226-230Crossref PubMed Scopus (335) Google Scholar, J. Neurosci. 2000; 20: PubMed Google Scholar). that were specific for the D1 and D2 receptors, we the and performed analysis of human and rat brain. In the was observed for both D1 and D2 receptors in cell of medium neurons corresponding to for both receptors could be which in cells expressing both D1 and D2 receptors. In the rat cells were shown to both D1 and D2 receptors, to only D2 receptors were for both receptors was co-localized in to at from the cell were also observed from the same that either D1 receptor or D2 receptor suggesting the possibility of at distinct of dopamine receptors within the same In cultures from rat D1 and D2 receptors were co-expressed in a significant of cells D2 receptor was observed in with both in the cell and the of of D1 receptors was distinct from that of D2 receptors in that was observed in only of and, in was to the cell and The high of of the used for D1 and D2 receptor was by of cells expressing each of the dopamine receptor of the receptors and with the tags were used to receptor expression in the and the was used to and of The D1 only cells expressing the D1 receptor and of the cells expressing the other dopamine receptors. was detected using the D2 in cells other than expressing the D2 receptor. Therefore, of the rat both and and of the human brain that there are but of D1 and D2 receptor This indicates that these two receptors are co-expressed within the same neurons and have the to with each of D1 and D2 have demonstrated that D1 and D2 receptors as in heterologous expression systems G. P. J. PubMed Scopus Google Scholar, T. 2003; PubMed Scopus Google Scholar) and in human and rat brain tissue P. T. P. Lett. PubMed Scopus Google Scholar). on the of dopamine receptors to we that an interaction between the D1 and D2 receptors may for the novel signal HA epitope-tagged D1 receptors and epitope-tagged D2 receptors were co-expressed in COS7 cells and using and HA were with a a corresponding to the D2 receptor was co-immunoprecipitation of receptor with was also In of from rat a corresponding to the D1 receptor was detected in proteins with a D1 receptor immunoprecipitation for the D2 receptor was performed and the was for the D1 receptor, the D1 receptor was detected indicating that D1 and D2 receptor were part of the same protein complex in rat In this study, we have the demonstration that two co-expressed receptors, when form a novel signaling complex. We have shown that agonist activation of co-expressed dopamine D1 and D2 receptors a PLC-mediated of intracellular calcium levels, a cellular response not with these receptors that have been shown to signal via the adenylyl cyclase The activation of a pathway by a complex of D1 and D2 receptors is an that these receptors are to signal by coupling to and The calcium signal was not observed stimulation of either receptor expressed alone or activation of cells co-expressing D1-D2 receptor with an agonist selective for only one of the receptors. has been that of the and receptors M. S. A. D.R. J.L. J. 20: PubMed Scopus Google Scholar) and of the and receptors T. R. G. J. 2000; PubMed Scopus Google Scholar) may to proteins distinct from with of receptors. However, the cellular response by these is not from that of the receptors intracellular for the receptors and of adenylyl cyclase for the results the demonstration of the coupling to a novel from the formation of a complex two distinct receptors. that neurons and within neurons were by D1 or D2 receptors and by D1 and D2 receptors, and cells that both receptors were to both stimulation and of adenylyl cyclase in to a calcium Therefore, it is that there are at of dopaminergic signaling by these two receptor The of a signaling pathway activated by D1 receptors may on or not the D1 receptors are with D2 receptors. The mechanism by which Gq to the D1-D2 heteromeric complex to be The direct interaction of Gq with the D1 receptor co-activation with the D2 receptor or Gq coupling to D2 receptor by co-activation with the D1 receptor are both there is evidence that only one protein is for each receptor of the receptor J.L. J. J. 2003; PubMed Scopus Google Scholar). A was to be the for on data that indicate that, the of proteins and only one protein could associate with a receptor to D. S. K. A. J. 2003; PubMed Scopus Google Scholar). studies a possibility in which protein may be by the of both the D1 and D2 receptors. calcium signaling by the D1 receptor has been shown to in some heterologous expression systems M. Pharmacol. 1995; Google Scholar, O. 1992; PubMed Scopus (41) Google Scholar, J. Jr., Greengard P. 1995; PubMed Scopus Google Scholar). This response is to and PKA, calcium be by and be blocked by In study, inhibitor treatment not the intracellular calcium this pathway as a mechanism of calcium activation by co-activated D1 and D2 receptors. between and receptors by has been observed to in potentiation of signaling to receptor activation U. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). A mechanism not to the novel signaling unit by co-activated D1 and D2 receptors, indicate that receptor to a Gq pathway in the of activation of both receptors. has also been demonstrated that a protein as with the D1 receptor to D1 receptor-mediated in intracellular calcium levels L. S. R. P. C. 2000; PubMed Scopus Google Scholar). However, the activation of the receptor complex by the activation of a co-expressed receptor, and is to be involved in the calcium signal in this was to a physiological basis for D1 and D2 receptor there has been considerable or not D1 and D2 receptors are co-localized within the same neurons (14Aizman O. Brismar H. Uhlen P. Zettergren E. Levey A.I. Forssberg H. Greengard P. Aperia A. Nat. Neurosci. 2000; 3: 226-230Crossref PubMed Scopus (335) Google Scholar, J. Neurosci. 2000; 20: PubMed Google Scholar). (14Aizman O. Brismar H. Uhlen P. Zettergren E. Levey A.I. Forssberg H. Greengard P. Aperia A. Nat. Neurosci. 2000; 3: 226-230Crossref PubMed Scopus (335) Google Scholar) have that, in the rat neurons expressing D1 subclass receptors also D2 subclass receptors. However, in studies have shown that D1 and D2 receptors not have in the striatum C.R. L.C. Monsma Jr., F.J. Sibley D.R. 1990; PubMed Scopus Google Scholar, A. R. H. O. Jr., 1991; Google Scholar, J. Neurosci. PubMed Google Scholar). highly specific D1 and D2 receptor and of human rat and neurons from rat we have shown that these two receptors were co-expressed and co-localized within a significant of In we have using inhibitors of signal that co-activated D1 and D2 receptors signal via a PLC-mediated calcium a pathway not activated by either receptor We have also shown D1 and D2 receptors are co-expressed and co-localized within neurons and that these two receptors are part of the same protein complex. studies have that these receptors were functionally linked. Therefore, a from studies of the D1 and D2 receptors, we present a for signaling by dopamine D1 and D2 receptors in where these two receptors function in a complex to intracellular calcium when The of from two receptors an of of receptor function be from a of receptors. Although these of a of not in of receptor it is that analysis of the function of receptor gene alone is and are of the of functional via may a of and by dopamine and other receptors. For there is a of evidence suggesting that of calcium signaling may be with the of in Brain Res. Brain Res. 2003; PubMed Scopus Google Scholar). In it be to this calcium signaling by co-activated D1 and D2 receptors is to and other

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