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• W2040624241 abstract "The expression and characteristics of the dopamine D3 receptor protein were studied in brain and in stably transfected GH3 cells. Monoclonal antibodies were used for immunoprecipitation and immunoblot experiments. Immunoprecipitates obtained from primate and rodent brain tissues contain a low molecular weight D3 protein and one or two larger protein species whose molecular mass are integral multiples of the low molecular weight protein and thus appear to have resulted from dimerization and tetramerization of a D3 monomer. Whereas D3receptor multimers were found to be abundantly expressed in brain, the major D3 immunoreactivity expressed in stable D3-expressing rat GH3 cells was found to be a monomer. However, multimeric D3 receptor species with electrophoretic mobilities similar to those expressed in brain were also seen in D3-expressing GH3 cells when a truncated D3-like protein (named D3nf) was co-expressed in these cells. Furthermore, results from immunoprecipitation experiments with D3- and D3nf-specific antibodies show that the higher-order D3 proteins extracted from brain and D3/D3nf double transfectants also contain D3nf immunoreactivity, and immunocytochemical studies show that the expression of D3 and D3nfimmunoreactivities overlaps substantially in monkey and rat cortical neurons. Altogether, these data show oligomeric D3 receptor protein expression in vivo and they suggest that at least some of these oligomers are heteroligomeric protein complexes containing D3 and the truncated D3nfprotein. The expression and characteristics of the dopamine D3 receptor protein were studied in brain and in stably transfected GH3 cells. Monoclonal antibodies were used for immunoprecipitation and immunoblot experiments. Immunoprecipitates obtained from primate and rodent brain tissues contain a low molecular weight D3 protein and one or two larger protein species whose molecular mass are integral multiples of the low molecular weight protein and thus appear to have resulted from dimerization and tetramerization of a D3 monomer. Whereas D3receptor multimers were found to be abundantly expressed in brain, the major D3 immunoreactivity expressed in stable D3-expressing rat GH3 cells was found to be a monomer. However, multimeric D3 receptor species with electrophoretic mobilities similar to those expressed in brain were also seen in D3-expressing GH3 cells when a truncated D3-like protein (named D3nf) was co-expressed in these cells. Furthermore, results from immunoprecipitation experiments with D3- and D3nf-specific antibodies show that the higher-order D3 proteins extracted from brain and D3/D3nf double transfectants also contain D3nf immunoreactivity, and immunocytochemical studies show that the expression of D3 and D3nfimmunoreactivities overlaps substantially in monkey and rat cortical neurons. Altogether, these data show oligomeric D3 receptor protein expression in vivo and they suggest that at least some of these oligomers are heteroligomeric protein complexes containing D3 and the truncated D3nfprotein. D3 receptors belong to the D2-class of dopamine receptors known to couple to inhibitory subsets of heterotrimeric G proteins. The members of the superfamily of G-protein-coupled receptors have a predicted membrane topology of seven hydrophobic transmembrane domains connected by alternating extra- and intracellular loops. Although it is generally thought that functional G-protein-coupled receptors contain a single receptor molecule, several recent observations suggest that these receptors also exists as oligomers. For example, muscarinic M2 receptor proteins purified from porcine atria contained, in addition to the monomer, multiples of the monomeric receptor with electrophoretic mobilities suggesting trimeric and tetrameric homoligomers (1Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Furthermore, the cooperative ligand binding profile of purified M2 receptors described by Wreggett and Wells (1Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) appears to fit best a model that assumes a tetrameric configuration of this receptor. Recently, Hebert et al. (2Hebert T.E. Moffett S. Morello J.-P. Loisel T.P. Bichet D.G. Barret C. Bovier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar) showed that β2-adrenergic receptors form homodimers in transfected cells. These homodimers were resistant to SDS and reducing agents. Most interestingly, this dimerization was found to be essential for β2-receptor-mediated stimulation of adenylyl cyclase, and agonist stimulation stabilized the dimeric receptor configuration, whereas inverse agonists favored the monomeric state (2Hebert T.E. Moffett S. Morello J.-P. Loisel T.P. Bichet D.G. Barret C. Bovier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar).Previous immunoblot and immunoprecipitation analyses of a variety of different G-protein-coupled receptors (expressed in insect or mammalian cells) also suggested that these receptors are capable of forming homoligomers. For example, dopamine D2 receptor immunoprecipitates contained two D2-immunoreactive protein species, the larger (∼93 kDa) being twice the size of the smaller (∼44 kDa) (3Ng G.Y.K. O'Dowd B.F. Caron M. Dennis M. Brann M.R. George S.R. J. Neurochem. 1994; 63: 1589-1595Crossref PubMed Scopus (102) Google Scholar). Similarly, dopamine D1 receptors were found to migrate at ∼50, 100, and 200 kDa (4Ng G.Y.K. Mouillac B. George S.R. Caron M. Dennis M. Bouvier M. O'Dowd B.F. Eur. J. Pharmacol. 1994; 267: 7-19Crossref PubMed Scopus (156) Google Scholar) on SDS-PAGE. 1The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.1The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline. In addition, oligomers of the m5-HT1b receptor (5Ng G.Y.K. George S.R. Zastawny R.L. Caron M. Bouvier M. Dennis M. O'Dowd B.F. Biochemistry. 1993; 32: 11727-11733Crossref PubMed Scopus (137) Google Scholar), the mGluR1 receptor (6Pickering D.S. Thomson C. Suzdak P.D. Fletcher E.J. Robitaille R. Salter M.W. MacDonald J.F. Huang X.P. Hampson D.R. J. Neurochem. 1993; 61: 85-92Crossref PubMed Scopus (100) Google Scholar), the substance P receptor (7Schreurs J. Yamamoto R. Lyons J. Munemitsu S. Conroy L. Clark R. Takeda Y. Krause J.E. Innis M. J. Neurochem. 1995; 64: 1622-1631Crossref PubMed Scopus (16) Google Scholar), the human C5a anaphylatoxin receptor (8Giannini E. Brouchon L. Boulay F. J. Biol. Chem. 1995; 270: 19166-19172Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and the platelet activating receptor (9Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar) were resolved under reducing conditions on SDS-PAGE. Although these results in cell lines were often interpreted as nonspecific aggregation of incompletely folded intermediates, in each case the higher molecular weight species appeared to comprise multiples of a monomer.Earlier studies on chimeric receptors already suggested the possibility that functional G-protein-coupled receptors can be comprised of multiple receptor molecules. For example, Maggio et al. (10Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar) made chimeric α2-adrenergic and M3 muscarinic receptors by replacing transmembrane domains VI and VII of one receptor with the corresponding domain of the other receptor. Although neither chimera was functional when expressed alone, functional receptors were formed when the two chimera were co-expressed. These results demonstrate that intermolecular interactions can occur between G-protein-coupled receptors and that the resultant oligomeric receptor complexes are functional.Whether G-protein-coupled receptors, in cells in which they are natively expressed, exist in monomeric or oligomeric configurations is still unresolved. However, the demonstration of functionaldifferences between dimers and monomers reported by Wreggett and Wells (1Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and Hebert et al. (2Hebert T.E. Moffett S. Morello J.-P. Loisel T.P. Bichet D.G. Barret C. Bovier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar) points to the importance of understanding the molecular composition of such receptors in vivo. The present study shows an oligomeric expression of the dopamine D3 receptor protein in brain which, at least in part, resulted from heteroligomerization of the D3protein and the truncated D3-like protein D3nf (see Refs. 11Schmauss C. Haroutunian V. Davis K.L. Davidson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8942-8946Crossref PubMed Scopus (132) Google Scholar and 12Liu K. Bergson C. Levenson R. Schmauss C. J. Biol. Chem. 1994; 269: 29220-29226Abstract Full Text PDF PubMed Google Scholar).RESULTSPolyclonal and monoclonal antibodies that were raised against peptide sequences specific for the human D3 receptor were used to characterize the properties and distribution of D3receptor species expressed in mammalian brain and in stably transfected rat GH3 cells.Characterization of the AntibodiesThe characterization of one of the D3 receptor antibodies used in this study has been reported previously (13Howe J.R. Skryabin B.V. Belcher S.M. Zerillo C.A. Schmauss C. J. Biol. Chem. 1995; 270: 14168-14174Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This antipeptide antibody, which was raised against the amino terminus of the human D3 receptor (Cambio, Cambridge, UK), detected D3 immunoreactivity in immunoblot experiments using proteins extracted from rat GH3 cells that stably express the human dopamine D3 receptor under the transcriptional control of a tetracycline-responsive promotor. The expression of D3 receptor mRNA and protein in these stably transfected cells is suppressed by including tetracycline in the culture medium and reaches steady-state levels 24 h after induction (13Howe J.R. Skryabin B.V. Belcher S.M. Zerillo C.A. Schmauss C. J. Biol. Chem. 1995; 270: 14168-14174Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Two novel monoclonal antibodies (IgM/D3 and IgG/D3), which were raised against peptide sequences constituting amino acid residues 252–284 that are part of the putative third cytoplasmic loop of the human D3 protein, were then tested for their ability to detect the same approximately 50-kDa D3-immunoreactivity recognized by the polyclonal antipeptide antibody raised against the amino terminus of the D3 protein. As shown in Fig.1 A, all three antibodies recognize the same protein of approximately 50 kDa, which appears on immunoblots of proteins extracted 5–9 h after induction of D3 expression in stably transfected GH3 cells. This D3 immunoreactivity reaches steady-state levels 24 h after the induction of expression and is not detected in non-transfected GH3 cells. The expression of this D3immunoreactivity is abolished 3 days after the inhibition of D3 mRNA expression by the addition of tetracycline (2 μg/ml) to the culture medium (Fig. 1 A).In addition, we synthesized a T7 RNA polymerase transcript of the human cDNA encoding the D3 receptor (cloned into the plasmid vector pRc/CMV; Invitrogen) and translated this mRNA in vitro in rabbit reticulocyte lysates. The resulting proteins were analyzed by immunoblotting using monoclonal antibodies IgM/D3 and IgG/D3 as well as the polyclonal anti-D3 antiserum. As shown in Fig. 1 B, all three antibodies recognized anin vitro translation product that also migrated at approximately 50 kDa. When D3 mRNA was omitted from thein vitro translation reaction, no D3immunoreactivity was detected on immunoblots, as shown in Fig.1 B for the polyclonal anti-D3 antiserum. It is noted in both experiments on transfected GH3 cells and onin vitro translation products that all three antibodies also recognize smaller protein species of about 37 and 30 kDa. These proteins are likely to be degradation products of the 50-kDa D3-immunoreactivity because they are not seen in non-transfected cells and they are also absent from in vitrotranslation reactions lacking D3 mRNA. Because these shorter protein species are also detected with the polyclonal antibody that recognizes amino-terminal D3 peptide sequences it is likely that these proteins represent D3 degradation products that lack carboxyl-terminal peptide sequences. The appearance of these putative degradation products is not due to insufficient supplementation of the SDS-containing solubilization buffer with protease inhibitors because they were detected in protein samples prepared in the presence of phenylmethylsulfonyl fluoride and aprotinin, and in samples that were additionally supplemented with pefabloc, pepstatin, and leupeptin (13Howe J.R. Skryabin B.V. Belcher S.M. Zerillo C.A. Schmauss C. J. Biol. Chem. 1995; 270: 14168-14174Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Altogether, the results shown in Fig. 1 demonstrate that all three antibodies (which were raised against non-overlapping peptide sequences of the human D3receptor) recognize the same D3 immunoreactivity.An additional polyclonal antibody that specifically recognizes a unique carboxyl-terminal peptide sequence contained only within the truncated D3-like protein named D3nf (11Schmauss C. Haroutunian V. Davis K.L. Davidson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8942-8946Crossref PubMed Scopus (132) Google Scholar), but not within the D3 protein, was used in some experiments. The characterization of this antibody has been reported previously (12Liu K. Bergson C. Levenson R. Schmauss C. J. Biol. Chem. 1994; 269: 29220-29226Abstract Full Text PDF PubMed Google Scholar).D3 Receptor Immunoreactivity Expressed in BrainThe monoclonal antibodies described above were used to characterize D3 immunoreactivity expressed in brain tissue. Because of the low levels of the D3 protein, immunoprecipitation experiments were performed using the monoclonal antibody (IgG/D3). The composition of the immunoprecipitate was analyzed on immunoblots using the monoclonal IgM/D3 antibody.We first analyzed the expression of D3 immunoreactivity expressed in human motor cortex. Fig.2 A shows the immunoprecipitates obtained with the IgG/D3 antibody. A protein of ∼50 kDa is clearly detected. However, an additional major D3 immunoreactivity of more than 180 kDa (∼200 kDa), and a smear of minor proteins migrating just below the largest protein, are also detected. The D3 immunoprecipitate of solubilized total human brain proteins (Fig. 2 A, lane 2) was then compared with the immunoprecipitate obtained from a membrane preparation of the same tissue (Fig. 2 A, lane 1; see “Experimental Procedures”). Also proteins solubilized from the membrane pellet contained the two major protein species indicating that both D3 protein species are integral membrane proteins. In lieu of the results shown in Fig. 1, the detection of the high molecular mass D3 immunoreactivity, which appears to be a multiple of the ∼50-kDa D3 protein, was unexpected. It should be noted, however, that even extensive post-translational modifications of the ∼50-kDa protein in vivo are unlikely to account for the mass of the additional large protein. For example, although dopamine receptors are thought to be extensivelyN-glycosylated, treatment of the D3immunoprecipitate with N-glycosidase F prior to blotting did not abolish the detection of the ∼200-kDa D3 protein (Fig. 2 A, lane 3). It did, however, abolish the smear of proteins seen directly below this protein species which revealed two sharp protein bands of slightly lower molecular mass. Furthermore, a doublet of proteins of ∼50 kDa is detected after treatment ofN-glycosidase F, indicating that removal ofN-linked sugars in a substantial portion of the D3 proteins results in only a small shift of their electrophoretic mobility.Figure 2D3-immunoreactivity expressed in human, monkey, and rat brain. A, D3immunoprecipitate of human motor cortical tissue. Lane 1, D3 immunoprecipitate obtained from 10 mg/ml protein of motor cortical membrane pellets. Lane 2, D3immunoprecipitate obtained from 6.5 mg/ml protein of total motor cortical tissue homogenate. Lane 3, the D3immunoprecipitate of motor cortical tissue protein was treated with 1 unit of glycosidase F prior to gel electrophoresis. B, D3 immunoprecipitate obtained from monkey and rat brain tissues. Lane 1, D3 immunoprecipitate of proteins extracted from monkey basal ganglia. Lane 2, D3 immunoprecipitate obtained from proteins extracted from rat prefrontal cortex. Proteins were immunoprecipitated with the monoclonal IgG antibody. C, D3immunoprecipitates of different rat tissues. Lane 1, prefrontal cortex; lane 2, rat muscle; lane 3, rat spleen. All proteins were immunoprecipitated with the monoclonal IgG/D3 antibody. The immunoblots of these precipitates were probed with the monoclonal IgM antibody. Bound antigens are visualized as described in the legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In further experiments, we tested the ability of the IgG/D3 antibody to immunoprecipitate the D3 protein expressed in monkey and rat brain. As shown in Fig. 2 B, the antibody also immunoprecipitated the D3 protein expressed in these two species, and in both species the protein composition of the immunoprecipitate is very similar. In addition to a ∼45-kDa protein, two larger proteins are contained in the immunoprecipitate that migrate at approximately 85 and 180 kDa. D3-immunoreactive signals were not detected when solubilized proteins from spleen or muscle tissue (which do not express D3 receptors) were incubated with the monoclonal IgG/D3 antibody (Fig. 2 C).The size of the 45-kDa D3 immunoreactivity obtained from monkey and rat brain tissue corresponds to the calculated molecular mass of the D3 core protein. Similar to the composition of the D3 immunoprecipitate obtained from human brain tissue, the two larger protein species appear to be multiples of the 45-kDa protein. The results, therefore, suggest that the D3protein may be expressed as a monomer, a dimer, or a tetramer in rodent and primate brain. Interestingly, in all three species examined the tetrameric configuration of the D3 protein is the most abundant protein species present in the immunoprecipitate. In fact, only the tetrameric, but not the dimeric, D3 protein configuration was detected in human brain (Fig. 2 A). In all three species, these higher-order structures of the D3receptor were shown to be resistant to reducing agents (thus suggesting that monomers are not linked to each other via disulfide bonds) and were maintained after exposure to SDS.D3 Receptor Immunoreactivity Expressed in Stably Transfected rat GH3 CellsThe results shown in Fig. 2 motivated us to examine in more detail whether higher-order structures of the D3 protein can also be detected in a single type of cell in which the level of D3 receptor expression can be controlled. We therefore re-examined the GH3 cells that stably and inducibly express D3 mRNA and protein. Fig.3 A (middle panel) shows the results of immunoblot experiments from three independent GH3 cell clones in which the expression of the D3 protein was maximally induced. In each of these clones, however, the major D3 immunoreactivity (detected by the monoclonal IgG/D3 antibody) is that of the approximately 50-kDa immunoreactivity also seen in Fig. 1. Some higher-order structures of the D3protein are also expressed albeit at very low levels. For example, low expression of an ∼90-kDa D3 immunoreactivity is detected and the largest band, although barely visible, migrates at about 180 kDa. (These faint bands are best seen in Fig. 3 B, last two lanes.) Thus, in contrast to the results we obtained with proteins extracted from brain tissue, the major D3 protein species detected in these cells appears to be a monomer. It should be noted that D3 protein expression has been characterized in a larger number of independent clones during various levels of induction, steady-state expression, or inhibition of expression. In all these clones we have never observed significant levels of expression of higher-order structures of the D3 protein. Interestingly, however, high levels of D3 immunoreactivity with an electrophoretic mobility of approximately 180 kDa was detected on immunoblots of proteins from cells that co-express the D3receptor and a truncated D3-like protein (named D3nf) which differs from the D3 protein only in its carboxyl-terminal peptide sequence and therefore is unable to form transmembrane spanning domains VI and VII (11Schmauss C. Haroutunian V. Davis K.L. Davidson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8942-8946Crossref PubMed Scopus (132) Google Scholar, 12Liu K. Bergson C. Levenson R. Schmauss C. J. Biol. Chem. 1994; 269: 29220-29226Abstract Full Text PDF PubMed Google Scholar). An immunoblot analysis of D3 protein expression in 5 randomly picked G-418- and hygromycin-resistant double-transfected GH3 cell clones (see “Experimental Procedures”) is shown in Fig. 3 A(right panel). Three of the 5 clones analyzed express, in addition to the 50-kDa D3 immunoreactivity also seen in single D3 transfectants, a major D3immunoreactivity migrating at approximately 180 kDa. Just as this result is different from the one obtained with single D3transfectants, it is also different from the results obtained with single D3nf transfectants in which the major D3nf immunoreactivity, recognized by a D3nf-specific polyclonal antiserum (12Liu K. Bergson C. Levenson R. Schmauss C. J. Biol. Chem. 1994; 269: 29220-29226Abstract Full Text PDF PubMed Google Scholar), migrates at approximately 45 kDa (Fig. 3 A, left panel).Figure 3Expression of D3 and D3nf immunoreactivities in stably transfected GH3 cells with active tetracycline-responsive promotors. A, immunoblots of proteins extracted from single D3nf and D3 transfectants, and from D3/D3nfdouble transfectants. All cells are in the fully induced state (i.e. steady-state protein expression). The blot on theleft, marked GH3/D3nf, was probed with the D3nf-specific antiserum (dilution 1:1000), the blots in themiddle, marked GH3/D3, and on theright, marked GH3/D3/D3nf, were probed with the D3-specific mAb (IgG, 1:100). Proteins were extracted from 3 (left and middle blots) or 5 (right blot) independent GH3 cell clones. Five μg of total cellular protein was loaded onto each lane. B, comparison of D3-immunoreactivities in D3/D3nfand D3/D2 double-transfectants, and in D3 single transfectants. These co-expressing cells were obtained after co-transfecting D3nf and D2, respectively, into the parent D3-expressing clone.Left blot, the first two lanes, markedD 3/D 3nf, contain proteins extracted from two independent clones with low (first lane) and high (second lane) levels of D3 and D3nf expression. The lanes markedD 3/D 2 andD 3 contain proteins extracted from cells that co-express D3 and D2 receptors and from cells that express only D3 receptors, respectively. Five μg of total cellular proteins were separated by SDS-PAGE and the blot was probed with the monoclonal IgG antibody. All cells are in the fully induced state. C, immunoprecipitation of proteins extracted from D3/D3nf double transfectants. Proteins extracted from the three double transfectants that express the 180-kDa D3 immunoreactivity were immunoprecipitated with the monoclonal IgG antibody. The immunoprecipitate was electrophoresed on SDS-PAGE. The blot was probed with the monoclonal IgM antibody (left). Bound antigens are visualized as described in the legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The expression of the 180-kDa D3 immunoreactivity does not appear to be the result of co-transfection per se, because it was not detected in D3-expressing GH3 cells that also express high mRNA levels of the (homologous) human D2 receptor (Fig.3 B, third lane). Furthermore, we observed the 180-kDa D3 immunoreactivity in a GH3 cell clone in which both D3 and D3nf are expressed at low levels (Fig.3 B, first lane) as well as in clones in which both proteins are expressed at very high levels (Fig. 3 B, second lane). However, in clones with high levels of D3, but very low levels of D3nf, the expression of the 180-kDa protein was not detected. This is, for example, the situation found for protein samples of the two G-418 and hygromycin-resistant GH3 clones loaded onto first and fifth lanes of Fig. 3 A(right panel). (Note that the gel shown in Fig.3 B is a 7% SDS-PAGE while the onces shown in Fig.3 A are 10 and 12% gels. This results in differences in the banding thickness of the 50-kDa protein.)In total, these results suggest that in GH3 cells the formation of the 180-kDa D3 immunoreactivity is specifically promoted by expression of the D3nf protein. The results also suggest that it is the molar ratio of the two proteins, and not their absolute level of expression, that determines the appearance of the higher-order D3 species; and the results do not therefore support the conclusion that the 180-kDa D3 immunoreactivity results from nonspecific protein aggregation due to overexpression of the transfected proteins (“molecular crowding”).In further experiments, proteins from the three double transfectants that express the 180-kDa D3 immunoreactivity were immunoprecipitated with the monoclonal IgG/D3 antibody and the protein composition was analyzed on immunoblots probed with the monoclonal IgM/D3 antibody (Fig. 3 C). As shown in Fig. 3 C, in each of the 3 clones, strong immunoreactive signals of approximately 180, 90, and 50 kDa were detected with monoclonal antibody IgM/D3. Thus, the pattern of D3 immunoreactivity contained in the immunoprecipitate of proteins from these D3/D3nf double transfectants is very similar to that seen in immunoprecipitates of proteins extracted from brain tissues (Fig. 2). It is noted, however, that the dimeric D3immunoreactivity is only detected in immunoprecipitation, but not immunoblotting, experiments on proteins of the GH3 double transfectants and that it is also not detected in immunoprecipitates of proteins extracted from human brain (Figs. 2 and 3). Although the reason for this discrepancy remains to be resolved, it is likely that the pellets of the immunoprecipitates contained mostly the 180-kDa protein complex which, during the preparation for SDS-PAGE analysis, could then be dissociated into its various protein constituents and that the detection of the dimeric D3 protein complex is therefore only possible when large amounts of the 180-kDa complex (i.e. those found in transfected cells, but not in brain tissue) are expressed.Protein Composition of the Higher-order D3Immunoreactivity in Brain and in D3/D3nf-expressing GH3 CellsOne possible explanation for the appearance of the ∼90- and 180-kDa protein species in co-transfected cells is that D3 and D3nf form heteroligomers. Both proteins have identical amino acid sequences that extend into the amino-terminal sequence of the putative third cytoplasmic domain, and thus include transmembrane spanning domains I through V. Although the D3nf protein is unlikely to have the additional transmembrane spanning domains VI to VII found in the D3 receptor protein, previous studies have shown molecular interactions between split G-protein-coupled receptor proteins (21Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (604) Google Scholar) or chimeric G-protein-coupled receptors (10Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar). The following experiments therefore tested whether the ∼85/90- and 180-kDa D3 immunoreactivities seen in immunoprecipitates of proteins extracted from D3/D3nf-expressing GH3 cells and, most importantly, from brain tissue also contain D3nf immunoreactivity.The results shown in Fig. 4 were obtained with proteins extracted from rat prefrontal cortex (lane 1) and from GH3 D3/D3nf double transfectants (lane 2) that were immunoprecipitated with the monoclonal IgG/D3 antibody. The immunoblot of these precipitates was probed with the D3nf-specific polyclonal antibody. Both the 180- and the ∼85/90-kDa protein species, but not the 50-kDa D3 monomer, gave D3nf-immunoreactive signals. A small fraction of the pellets of these immunoprecipitates (approximately 10%) was analyzed on immunoblots probed with the monoclonal IgM/D3 antibody. The results (not shown) are identical to the results shown in Figs. 2 and 3 C, e.g. all three D3-immunoreactive protein species, including the ∼50-kDa monomer, were detected. In addition, proteins extracted from the rat brain were immunoprecipitated with the D3nf-specific polyclonal antiserum and the immunoblot of this precipitate was probed with the monoclonal IgM/D3 antibody. As shown in Fig. 4 (lane 3), both the higher-order D3 species, but not the 45-kDa D3nf immunoreactivity seen in single D3nf transfectants (see Fig. 3 A), are also recognized by the D3-specific antibody. (In general, however, we found the D3nf antiserum less" @default.
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• W2040624241 title "Expression of Dopamine D3 Receptor Dimers and Tetramers in Brain and in Transfected Cells" @default.
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