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• W2000658108 abstract "Based on its expression pattern and pharmacology, the D4 dopamine receptor may play a role in schizophrenia. Thus it is of interest to know what signaling pathways are utilized by this receptor. Previously, we showed that activation of D4 receptors in a mouse mesencephalic neuronal cell line (MN9D) inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive (Ptx-sensitive) fashion. Of the known Ptx-sensitive G-protein α subunits, MN9D-expressed Gαi2, GαoA, and GαoB; however, none of these coupled to the D4 receptor. Using a low stringency polymerase chain reaction cloning method, we found an additional Ptx-sensitive G-protein cone transducin (Gαt2) expressed in the MN9D cells. We also found that Gαt2 mRNA is highly expressed in rat mesencephalic tissue. To test the hypothesis that the D4 receptor couples to Gαt2, we cotransfected MN9D cells with the D4 receptor and a mutagenized Ptx-resistant Gαt2 subunit (mGαt2). Application of the dopaminergic agonist quinpirole to cotransfected cells inhibited forskolin-stimulated cAMP accumulation in the presence or absence of Ptx. To our knowledge, this is the first report demonstrating that the D4 dopamine receptor functionally couples to a specific G-protein and that a non-opsin-like receptor can couple with a transducin subunit. Based on its expression pattern and pharmacology, the D4 dopamine receptor may play a role in schizophrenia. Thus it is of interest to know what signaling pathways are utilized by this receptor. Previously, we showed that activation of D4 receptors in a mouse mesencephalic neuronal cell line (MN9D) inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive (Ptx-sensitive) fashion. Of the known Ptx-sensitive G-protein α subunits, MN9D-expressed Gαi2, GαoA, and GαoB; however, none of these coupled to the D4 receptor. Using a low stringency polymerase chain reaction cloning method, we found an additional Ptx-sensitive G-protein cone transducin (Gαt2) expressed in the MN9D cells. We also found that Gαt2 mRNA is highly expressed in rat mesencephalic tissue. To test the hypothesis that the D4 receptor couples to Gαt2, we cotransfected MN9D cells with the D4 receptor and a mutagenized Ptx-resistant Gαt2 subunit (mGαt2). Application of the dopaminergic agonist quinpirole to cotransfected cells inhibited forskolin-stimulated cAMP accumulation in the presence or absence of Ptx. To our knowledge, this is the first report demonstrating that the D4 dopamine receptor functionally couples to a specific G-protein and that a non-opsin-like receptor can couple with a transducin subunit. Dopamine is a major neurotransmitter in a wide variety of organisms. In mammalian brain, dopamine modulates motor, affective, cognitive, and neuroendocrine functions. There are five cloned dopamine receptors, D1–5, that can be distinguished by pharmacological and physiological criteria (1Kebabian J.W. Calne D.B. Nature. 1979; 277: 93-96Crossref PubMed Scopus (3149) Google Scholar, 2Zhou Q.-Y. Grandy D.K. Thambi L. Kushner J.A. Van Tol H.H.M. Cone R. Pribnow D. Salon J. Bunzow J.R. Civelli O. Nature. 1990; 347: 76-80Crossref PubMed Scopus (509) Google Scholar, 3Sunahara R.K. Guan H.C. O'Dowd B.F. Seeman P. Laurier L.G. Ng G. George S.R. Torchia J. Van Tol H.H. Niznik H.B. Nature. 1991; 350: 614-619Crossref PubMed Scopus (942) Google Scholar, 4Sunahara R.K. Niznik H.B. Weiner D.M. Storman T.M. Brann M.R. Kennedy J.L. Gelernter J.E. Rozmahel R. Yang Y. Israel Y. Seeman P. O'Dowd B.F. Nature. 1990; 347: 80-83Crossref PubMed Scopus (399) Google Scholar, 5Bunzow J.R. Van Tol H.H.M. Grandy D.K. Albert P. Salon J. Christie M. Machida C.A. Neve K.A. Civelli O. Nature. 1988; 336: 783-787Crossref PubMed Scopus (976) Google Scholar, 6O'Malley K.L. Harmon S. Tang L. Todd R.D. New Biol. 1992; 4: 137-146PubMed Google Scholar, 7Sokoloff P. Giros B. Martre M.-P. Bouthenet M.-L. Schwartz J.-C. Nature. 1990; 347: 146-151Crossref PubMed Scopus (2425) Google Scholar, 8Van Tol H.H.M. Bunzow J.R. Guan H.-C. Sunahara R.K. Seeman P. Niznik H.B. Civelli O. Nature. 1991; 350: 610-614Crossref PubMed Scopus (1862) Google Scholar). Of these, the D4 receptor has engendered much interest due to its possible involvement in schizophrenia. The D4 receptor exhibits a high affinity for clozapine, the prototype for a new class of antipsychotic drugs with reduced extrapyramidal side effects (8Van Tol H.H.M. Bunzow J.R. Guan H.-C. Sunahara R.K. Seeman P. Niznik H.B. Civelli O. Nature. 1991; 350: 610-614Crossref PubMed Scopus (1862) Google Scholar). This is in keeping with its distribution in limbic and cortical brain regions as well as its relative absence in striatal regions (6O'Malley K.L. Harmon S. Tang L. Todd R.D. New Biol. 1992; 4: 137-146PubMed Google Scholar, 8Van Tol H.H.M. Bunzow J.R. Guan H.-C. Sunahara R.K. Seeman P. Niznik H.B. Civelli O. Nature. 1991; 350: 610-614Crossref PubMed Scopus (1862) Google Scholar, 9Mrzljak L. Bergson C. Pappy M. Huff R. Levenson R. Goldman-Rakic P.S. Nature. 1996; 381: 245-248Crossref PubMed Scopus (428) Google Scholar). Studies have also shown that D4 receptors may be increased 2-fold in schizophrenic brain (10Seeman P. Guan H.-C. Van Tol H.H.M. Nature. 1993; 365: 441-445Crossref PubMed Scopus (616) Google Scholar). In rat striatum, treatment for 1 month with the antipsychotic haloperidol elevates the density of D4 receptors by about 2-fold, whereas D2 and D3 are only modestly affected (11Schoots O. Seeman P. Guan H.-C. Paterson A.D. Van Tol H.H.M. Eur. J. Pharmacol. 1995; 289: 67-72Crossref PubMed Scopus (54) Google Scholar). Characterization of the signal transduction pathways of D4and other dopamine receptors in vivo has been difficult because of the heterogeneity of tissue and cell types as well as the lack of truly selective agonists and antagonists. Transiently or stably expressed D4 receptors in several cell lines show similar binding characteristics and effects of guanine nucleotides on agonist binding (8Van Tol H.H.M. Bunzow J.R. Guan H.-C. Sunahara R.K. Seeman P. Niznik H.B. Civelli O. Nature. 1991; 350: 610-614Crossref PubMed Scopus (1862) Google Scholar, 12Chabert C. Cavegn C. Bernard A. Mills A. J. Neurochem. 1994; 63: 62-65Crossref PubMed Scopus (36) Google Scholar, 13Mills A. Allet B. Bernard A. Chabert C. Brandt B. Cavegn C. Chollet A. Kawashima E. FEBS Lett. 1993; 320: 130-134Crossref PubMed Scopus (49) Google Scholar, 14McHale M. Coldwell M.C. Herrity N. Boyfield I. Winn F.M. Ball S. Cook T. Robinson J.H. Gloger I.S. FEBS Lett. 1994; 345: 147-150Crossref PubMed Scopus (20) Google Scholar, 15Chio C.L. Drong R.F. Riley D.T. Gill G.S. Slightom J.L. Huff R.M. J. Biol. Chem. 1994; 269: 11813-11819Abstract Full Text PDF PubMed Google Scholar). For example, in HEK and CHO cells transfected with the D4 receptor, forskolin-stimulated cAMP accumulation is reduced by the D2, D3, and D4receptor agonist, quinpirole (8Van Tol H.H.M. Bunzow J.R. Guan H.-C. Sunahara R.K. Seeman P. Niznik H.B. Civelli O. Nature. 1991; 350: 610-614Crossref PubMed Scopus (1862) Google Scholar, 14McHale M. Coldwell M.C. Herrity N. Boyfield I. Winn F.M. Ball S. Cook T. Robinson J.H. Gloger I.S. FEBS Lett. 1994; 345: 147-150Crossref PubMed Scopus (20) Google Scholar, 15Chio C.L. Drong R.F. Riley D.T. Gill G.S. Slightom J.L. Huff R.M. J. Biol. Chem. 1994; 269: 11813-11819Abstract Full Text PDF PubMed Google Scholar). Previous studies in our laboratory found that D4 receptors are present in the photoreceptor layer of mouse retina and that dopaminergic agonists reduce light-sensitive cAMP levels with a D4-like pharmacological profile (16Cohen A.I. Todd R.D. Harmon S. O'Malley K.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12093-12097Crossref PubMed Scopus (185) Google Scholar). We also demonstrated that D4 receptors inhibited forskolin-stimulated cAMP accumulation in a dopaminergic cell line, MN9D (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar, 18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). To identify the G-protein(s) that couples to the D4 receptor, MN9D cells were cotransfected with D4 and a pertussis toxin-resistant (Ptx-resistant) 1The abbreviations used are: Ptx, pertussis toxin; PCR, polymerase chain reaction; mGαt2, mutagenized Ptx-resistant Gαt2 subunit; RT, reverse transcriptase; PDE, phosphodiesterase; IBMX, 3-isobutyl1-methylxanthine. mutant of Gαi2, Gαi3, GαoA (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar), or GαoB. 2C. O'Hara, R. D. Todd, and K. L. O'Malley, unpublished data. However, none of the G-protein α subunits tested could rescue receptor-mediated inhibition of forskolin-stimulated cAMP accumulation from Ptx treatment. Based on this result, we attempted to clone additional G-protein α subunits from these cells using PCR-based strategies. Here we show that cone transducin (Gαt2) is expressed in MN9D cells and demonstrate that it couples to the D4 receptor. All enzymes were purchased from New England Biolabs Inc. (Beverly, MA) or Promega (Madison, WI). The expression vectors pcDNA1/neo and pHook-2 were obtained from Invitrogen (San Diego, CA). Eticlopride, spiperone, (+)-butaclamol, (−)-quinpirole, forskolin, and pertussis toxin were from Research Biochemical Inc. (Wayland, MA). G418, Dulbecco's modified Eagle's medium, fetal calf serum, and LipofectAMINE reagent were from Life Technologies, Inc. (Gaithersburg, MD). [3H]Spiperone was purchased from NEN Life Science Products. Four degenerate primers were designed from conserved regions of the G-protein α subunit family, Gαi, Gαo, and Gαt. These included amino acids STIVKQM (o-865, 5′-AG(C/T)ACIAT(C/T)GT(G/C)AA(A/G)CAGATGAA-3′), KKWIHCF (o-967, 5′-AA(A/G)AA(A/G)TGGATCCACTGCTT(C/T)GA-3′), NPKDCGLF (o-965, 5′-A(G/A)AA(G/A)AG(C/G)CC(A/G)CAGTC(T/C)TT(C/G)AGGTT-3′), and HMTCATD (o-966, 5′-GTGTCIGT(G/A)GC(G/A)CAIGTCATGTG-3′) (19Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (548) Google Scholar). Oligonucleotides made to NPKDCGLF (o-965) and HMTCATD (o-966) were in the antisense orientation. Total MN9D RNA was reverse-transcribed using primer o-965 and then amplified using primers o-865 and o-965 (94 °C, 1 min; 50 °C, 2 min; 72 °C, 3 min; 30 cycles). First round PCR products were similarly amplified using the internal primers o-966 and o-967. The isolated PCR fragments were subcloned into plasmid pCR II (Invitrogen) and sequenced (Sequenase version 2.0 DNA sequencing kit, Amersham Life Science, Inc.). Total RNA was isolated from various tissues and MN9D cells as described (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Gαt2-specific messages (21Kubo M. Hirano T. Kakinuma M. FEBS Lett. 1991; 291: 245-248Crossref PubMed Scopus (12) Google Scholar) were detected using primers o-955 (5′-TTCTCTAGAGCTGGAGAAGAAGCTG-3′, identical to nucleotides 58–74) and o-978 (5′-CTAGAGTGGACATGGCTC-3′, complementary to nucleotides 262–278) at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s for 30 cycles. In other experiments, primers o-955 and o-956 (5′-TATTCTAGATGGCCAGGATGGACTG-3′, complementary to nucleotides 241–258) were used. Products were electrophoresed, transferred to a nylon membrane, and hybridized with a radiolabeled internal primer o-1053 (5′-ATTGTCAAACAGATGAAGAT-3′). Image analysis was performed using a PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). The mRNA levels were normalized for equal amounts of the 18 S fragment of ribosomal RNA (22Chan Y.L. Gutell R. Noller H.F. Wool I.G. J. Biol. Chem. 1984; 259: 224-230Abstract Full Text PDF PubMed Google Scholar) as described (6O'Malley K.L. Harmon S. Tang L. Todd R.D. New Biol. 1992; 4: 137-146PubMed Google Scholar). An expression vector containing the bovine Gαt2 cDNA was kindly provided by N. Gautam (Washington University School of Medicine, St. Louis, MO). A Ptx-resistant Gαt2 clone was constructed by introducing a cysteine-to-serine change four residues from the carboxyl terminus (23Taussig R. Sanchez S. Rifo M. Gilman A.G. Belardetti F. Neuron. 1992; 8: 799-809Abstract Full Text PDF PubMed Scopus (107) Google Scholar) using standard techniques for site-directed mutagenesis (24Higuchi R. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA1990: 177-183Google Scholar) and the primers o-983 (5′-ACCTCAA(A/G)GACAGCGG(A/G)CTCTTCT-3′, nucleotides 1045–1066) and its complement, o-984 (25Lerea C.L. Somers D.E. Hurley J.B. Klock I.B. Bunt-Milan A.H. Science. 1986; 234: 77-80Crossref PubMed Scopus (171) Google Scholar). The resulting clone (mGαt2) was confirmed by sequence analysis. MN9D cells (26Choi H.K. Won L.A. Kontur P.J. Hammond D.N. Fox A.P. Wainer B.H. Hoffman P.C. Heller A. Brain Res. 1991; 552: 67-76Crossref PubMed Scopus (200) Google Scholar, 27Choi H.K. Won L. Roback J.D. Wainer B.H. Heller A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8943-8947Crossref PubMed Scopus (86) Google Scholar) were maintained and transfected as described previously (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar, 18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). Each stable subclone was screened for the rat D4 receptor and mGαt2 mRNA expression by RT-PCR assays. Several rounds of further subcloning by limiting dilution were performed to establish homogeneous cell populations. Membrane preparation, receptor binding assays, and cellular cAMP measurements were performed exactly as described (18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). Saturation binding data were analyzed using programs from Lundon Software (Chagrin Falls, OH). For Ptx treatment, cells were incubated overnight in 50 ng/ml Ptx, which was previously shown to be a maximally effective dose (18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). Experiments using the Capture Tec pHook-2 kit (Invitrogen) were performed according to manufacturer protocol. pHook-2 encodes a fusion protein consisting of a signal peptide, a single chain antibody against the hapten, phOx, and a transmembrane domain to anchor the antibody in the plasma membrane (28Hoogenboom H.R. Griffiths A.D. Johnson K.S. Chriswell D.J. Hudson P. Winter G. Nucleic Acids Res. 1991; 19: 4133-4137Crossref PubMed Scopus (896) Google Scholar, 29Griffin G.M. Berek C. Kaartinen M. Milstein C. Nature. 1984; 312: 271-275Crossref PubMed Scopus (429) Google Scholar, 30Coloma M.J. Hastings A. Wims L.A. Morrison S.L. J. Immunol. Methods. 1992; 152: 89-104Crossref PubMed Scopus (163) Google Scholar). pHook-2-expressing cells can be isolated from whole cultures magnetically using beads coated with phOx. All data were normally distributed. For multiple comparisons within a given cell line, one-way analysis of variance was used to estimate significance followed by post hoc t tests corrected for multiple comparisons by the method of Bonferroni. For the single comparisons using a single cell line, pairedt tests were used. All analyses were adjusted for inequality of variances when appropriate. All analyses were completed using the SAS suite of programs (SAS Institute Inc., Cary, NC). Previously, we showed that the G-protein α subunits Gαi2 and GαoA that are expressed in MN9D cells (18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar) do not couple to D4 receptors in order to decrease cAMP levels (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar). Similar studies have also ruled out coupling to Gαi3(17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar), Gαi1, and GαoB. 2C. O'Hara, R. D. Todd, and K. L. O'Malley, unpublished data. To determine whether other known or novel Gα subunits were present in this cell line, we used a low stringency nested PCR approach. Sets of degenerate primers derived from conserved domains among the Ptx-sensitive Gα subunits were synthesized and used to amplify cDNA derived from MN9D cells. PCR products were subcloned and transformed then randomly selected. Clones were further identified by sequence analysis. As shown in Table I, Gαi2 and GαoAsequences were recovered (18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar) as well as a single Gαi3clone. No Gαi1 or Gαt1 clones were detected in this screen (not shown). Unexpectedly, 40% of the clones sequenced corresponded to mouse Gαt2. To verify that Gαt2 was expressed in MN9D cells, we used mouse-specific primer pairs to amplify Gαt2 mRNA. The predicted Gαt2 band was seen at a level comparable to that present in mouse retina (Fig. 1 A).Table IResults of screening MN9D cells with degenerate oligonucleotides from conserved Gαi, Gαo and GαtdomainsPrimersGαoA/GαoBGαi2Gαi3Gαt2865/967011115976/9771201955/9561000952/9532200The G-protein subtypes were identified by sequence analysis. The primer pairs used would not have distinguished GαoA or GαoB. Open table in a new tab The G-protein subtypes were identified by sequence analysis. The primer pairs used would not have distinguished GαoA or GαoB. Because MN9D cells were derived from dopaminergic mesencephalic cells (26Choi H.K. Won L.A. Kontur P.J. Hammond D.N. Fox A.P. Wainer B.H. Hoffman P.C. Heller A. Brain Res. 1991; 552: 67-76Crossref PubMed Scopus (200) Google Scholar, 27Choi H.K. Won L. Roback J.D. Wainer B.H. Heller A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8943-8947Crossref PubMed Scopus (86) Google Scholar), we examined mesencephalic tissue as well as other central nervous system and peripheral tissues for the presence and relative abundance of Gαt2 transcripts. As suggested by its presence in MN9D cells, Gαt2 mRNA was abundant in the mesencephalon as well as in the retina (Fig. 1 B). Moreover, detectable but relatively low levels of Gαt2 transcripts were seen in other central nervous system and peripheral tissues, with the exception of the kidney, where Gαt2 mRNA was more abundantly expressed. Taken together, these data suggest that Gαt2is expressed outside of the retina, where it may couple to non-opsin-like receptors. To test the hypothesis that D4 receptors can couple to Gαt2, we cotransfected MN9D cells with the rat D4 receptor and a Ptx-resistant mGαt2. In this paradigm, Gαt2 has been mutated such that the expressed protein is incapable of being ADP-ribosylated, thus rendering it insensitive to Ptx (23Taussig R. Sanchez S. Rifo M. Gilman A.G. Belardetti F. Neuron. 1992; 8: 799-809Abstract Full Text PDF PubMed Scopus (107) Google Scholar). If D4 receptors couple to Gαt2 to inhibit cAMP accumulation, then cotransfection with the mutagenized Gαt2 should rescue the receptor-stimulated inhibition of cAMP accumulation from Ptx treatment. Cotransfected, stable cell lines (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar) were characterized by RT-PCR to confirm D4 receptor expression (not shown) and Gαt2 (Fig. 2 A). Because the primers chosen were from the bovine Gαt2 3′-flanking region, the endogenous mouse cone message would not have been detected. Next, we used an indirect approach to look for mGαt2protein expression because we were unable to obtain antibodies directed against bovine Gαt2. Since Gαt2 cDNA was expressed together with the fusion protein in the pHook vector system, we measured expression of the pHook cell surface antibody. As shown in Fig. 2 B, when magnetic beads coated with phOx hapten were added to cell cultures, the D4 receptor cells transfected with the Gαt2/phOx vector were surrounded by beads, whereas parental cells were not. Finally, the level of D4 receptor expression was determined by saturation binding using increasing concentrations of [3H]spiperone, a D2-like dopamine receptor antagonist, and (±)-butaclamol to define nonspecific binding. [3H]Spiperone bound to MN9D-D4/mGαt2 cell membranes in a saturable fashion with a B max of 3.42 ± 0.36 pmol/mg of protein (mean ± S.D., n = 3) and aK d of 0.56 ± 0.40 nm (mean ± S.D., n = 3) (Fig. 2 C). No specific [3H]spiperone binding was detected in untransfected MN9D cells (18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). Previously, we showed that agonist stimulation of D4 receptors in MN9D cells inhibited forskolin-stimulated cAMP accumulation by 30–40% (17O'Hara C.M. Tang L. Taussig R. Todd R.D. O'Malley K.L. J. Pharmacol. Exp. Ther. 1996; 278: 354-360PubMed Google Scholar, 18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). Fig.3 shows comparable and significant inhibition of cAMP accumulation in cells with or without Gαt2. D4-mediated inhibition of cAMP accumulation could be blocked by overnight pretreatment with 50 ng/ml Ptx (Fig. 3 Aand Ref. 18Tang L. Todd R.D. Heller A. O'Malley K.L. J. Pharmacol. Exp. Ther. 1994; 268: 495-502PubMed Google Scholar). In contrast, in the cotransfected MN9D-D4/mGαt2 cells, Ptx could no longer block the effects of agonist treatment (Fig. 3 B). Thus the D4 receptor can couple to Gαt2, making it, to our knowledge, the first non-opsin-like receptor to couple to this retinal-enriched G-protein. Transducins are known to couple to cyclic nucleotide phosphodiesterases (PDEs) leading to decreased cyclic nucleotide levels and closure of cation-specific channels in the plasma membrane (31Stryer L. J. Biol. Chem. 1991; 266: 10711-10714Abstract Full Text PDF PubMed Google Scholar). To test the hypothesis that D4-mediated inhibition of cAMP levels is achieved via coupling to Gαt2 followed by activation of a cAMP PDE, we performed cAMP accumulation assays in the presence of several PDE inhibitors. We first tested 1 mm3-isobutyl-1-methylxanthine (IBMX), a non-selective PDE inhibitor. However, IBMX did not block the inhibition of forskolin-stimulated cAMP accumulation by quinpirole (n = 4) nor did 8-methoxymethyl-IBMX (25 μm), a specific inhibitor of PDE type I, trequinsin (10 nm), a PDE type III specific inhibitor, rolipram (10 μm), a type IV inhibitor, or a type V inhibitor (4-{[3′,4′-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline, 1 μm). We have also looked for but not found any changes in cGMP levels associated with receptor stimulation. Thus the downstream target of D4 receptor-activated Gαt2 (or its associated βγ dimer) in MN9D cells is unclear at present. PCR-based cloning techniques allowed us to survey the repertoire of Ptx-sensitive G-protein α subunits in the mesencephalic-derived MN9D cell line (26Choi H.K. Won L.A. Kontur P.J. Hammond D.N. Fox A.P. Wainer B.H. Hoffman P.C. Heller A. Brain Res. 1991; 552: 67-76Crossref PubMed Scopus (200) Google Scholar, 27Choi H.K. Won L. Roback J.D. Wainer B.H. Heller A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8943-8947Crossref PubMed Scopus (86) Google Scholar). Gαt2 was isolated with about the same frequency as Gαi2 using several different primer sets. Gαt2 was also expressed in nontransfected mesencephalic tissue as well as other central nervous system and peripheral tissue. Cellular expression of a Ptx-resistant mutant of Gαt2 restored agonist-stimulated inhibition of cAMP accumulation in D4-expressing cells in which the endogenous G-proteins were uncoupled from the receptor by pretreatment with Ptx. This is the first report of a non-opsin-like receptor coupling to a transducin as well as the first identification of a specific G-protein coupled to the D4 dopamine receptor. Previously, we have shown that the D4 receptor is most abundantly expressed in the inner segment layer of mouse photoreceptors (16Cohen A.I. Todd R.D. Harmon S. O'Malley K.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12093-12097Crossref PubMed Scopus (185) Google Scholar). Moreover, in this tissue, the light-sensitive pool of cAMP can be eliminated in the dark by application of ligands with the rank order of affinities characteristic of the D4 receptor. Thus,in vivo the D4 receptor is physiologically coupled to the modulation of cAMP levels. Our finding that the D4 receptor can couple to Gαt2 in transfected cells raises the possibility that this also occurs within the retina. Numerous studies have suggested that Gαt2 is specifically expressed in the retina (25Lerea C.L. Somers D.E. Hurley J.B. Klock I.B. Bunt-Milan A.H. Science. 1986; 234: 77-80Crossref PubMed Scopus (171) Google Scholar). Recently, however, Zigman et al. (32Zigman J.M. Westermark G.T. Lamendola J. Steiner D.F. Endocrinology. 1994; 135: 31-37Crossref PubMed Google Scholar) found Gαt2 mRNA in pancreatic islet cells, adrenal gland, and pituitary. The presence of Gαt2in immortalized cells from the central nervous system as well as in various central nervous system and peripheral tissues confirms and extends these observations. Interestingly, the D4 receptor is expressed in some but not all of these regions. For example in rats, D4 receptors are present in kidney (33Sun D. Schafer J.A. Am. J. Physiol. 1996; 271: F391-F400Crossref PubMed Google Scholar) and retina (16Cohen A.I. Todd R.D. Harmon S. O'Malley K.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12093-12097Crossref PubMed Scopus (185) Google Scholar) but not in mesencephalic tissues (6O'Malley K.L. Harmon S. Tang L. Todd R.D. New Biol. 1992; 4: 137-146PubMed Google Scholar). Given the high level of Gαt2 in the latter region (Fig. 1 B), this suggests that Gαt2 can couple to other receptors as well. What is the function of Gαt2 outside of the retina? Within photoreceptors, Gαt1 and Gαt2activate cGMP PDE, leading to decreased cGMP levels and closure of cation-specific channels in the plasma membrane (31Stryer L. J. Biol. Chem. 1991; 266: 10711-10714Abstract Full Text PDF PubMed Google Scholar). However, recent studies by Margolskee and co-workers (34Ruiz-Avila L. Mclaughlin S.K. Wildman D. Mckinnon P.J. Robichon A. Spickofsky N. Margolskee R.F. Nature. 1995; 376: 80-85Crossref PubMed Scopus (177) Google Scholar, 35Kolesnikov S.S. Margolskee R.F. Nature. 1995; 376: 85-88Crossref PubMed Scopus (100) Google Scholar) have shown that rod transducin is also present in vertebrate taste cells, where it appears to activate a cAMP-specific PDE. In this cell type, phosphodiesterase-mediated degradation of cAMP appears to activate a cyclic nucleotide-suppressive channel, leading to depolarization and Ca2+ influx (35Kolesnikov S.S. Margolskee R.F. Nature. 1995; 376: 85-88Crossref PubMed Scopus (100) Google Scholar). Thus, rod transducin also appears to be functionally important outside of the retina, although the receptor to which it is coupling has yet to be identified. We tested the hypothesis that D4-mediated inhibition of cAMP accumulation is achieved via coupling to Gαt2followed by activation of a cAMP PDE by performing cAMP accumulation assays in the presence of several PDE inhibitors. To date, however, none of the PDE inhibitors tested appeared to have any effect. We have also looked for but not found any changes in cGMP levels associated with receptor stimulation. Conceivably, the β/γ subunits associated with Gαt2 might modulate adenylyl cyclase activity, as observed with β/γ subunits associated with Gαi or Gαo (36Tang W.-J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (748) Google Scholar). Alternatively, IBMX-insensitive PDEs might be involved. Further investigations are needed to determine what the downstream signaling molecule is in MN9D cells. Although D1 and D2 dopamine receptors appear to mediate most of the known biological effects of dopamine receptors in the central nervous system, the presence and expression patterns of the less abundantly expressed receptors such as D4 make their function all the more intriguing. The mechanisms mediating the effects of D4 are of particular interest. Since the D4receptor may be involved in schizophrenia, knowledge of its signal transduction pathway might be of therapeutic importance as well. We gratefully acknowledge Drs. A. Heller, L. Won, and P. Hoffman not only for providing us with the MN9D cell line but also for their many insightful comments in using this system. We also thank Drs. N. Gautam and R. Reed for insightful discussions and J. M. Hickok for technical and statistical support." @default.
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• W2000658108 cites W2067346266 @default.
• W2000658108 cites W2068438393 @default.
• W2000658108 cites W2073768420 @default.
• W2000658108 cites W2075028631 @default.
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