Chimeric D1/D2 dopamine receptors. Distinct determinants of selective efficacy, potency, and signal transduction.
D1/D2 chimeras were constructed that had D1 dopamine receptor sequence at the amino-terminal end and D2 dopamine receptor sequence at the carboxyl-terminal end. The chimeras with the first four, five and six transmembrane domains of the D1 receptor (CH2, CH3, CH4, respectively) bound the D1 receptor antagonist [3H]SCH 23390 with high affinity. Reciprocal chimeras constructed with D2 receptor sequence at the amino-terminal end displayed no detectable specific binding of [3H]SCH 23390, [125I]epidepride, or [3H]spiperone. CH2, CH3, and CH4 had lower affinity than either D1 or D2 dopamine receptors for the nonselective antagonists and agonists and D2-selective antagonists tested. The chimeric receptors had affinities for three D1-selective ligands and the D2-selective agonist, quinpirole, that were intermediate between D1 and D2 receptor affinities for the drugs. The substantial loss or gain of affinity for three ligands upon replacement of D1 transmembrane VII with D2 sequence (CH4) suggests an important role for this region in the selectivity of these drugs. Stimulation of adenylyl cyclase activity by D1 agonists occurred in cells expressing CH3 and CH4, both of which included the D1 third cytoplasmic loop, but not in cells expressing CH1 or CH2, both with the D2 third cytoplasmic loop. However, only CH3 was able to mediate stimulation of adenylyl cyclase by quinpirole, implying that D2 receptor transmembrane domain VI was an important determinant of the selective efficacy of quinpirole. On the other hand, transmembrane domain VII was particularly important for the selective potency of quinpirole. Inhibition of beta-adrenergic receptor-stimulated adenylyl cyclase activity by dopamine was seen in cells expressing D2 receptors and CH1, but not CH2, CH3, or CH4. Thus, the third cytoplasmic loop of D1 dopamine receptors was crucial for the coupling of the receptors to Gs, but inhibition of adenylyl cyclase via Gi required structural features, such as the second cytoplasmic loop of the D2 receptor, in addition to the 3rd cytoplasmic loop.
Kozell
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LB
Veterans Affairs Medical Center, Portland, Oregon 97201.
Machida
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Neve
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Neve
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism; D001552:Benzazepines / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D017931:DNA Primers; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D018491:Dopamine Agonists / Q000378:metabolism / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000378:metabolism / Q000494:pharmacology; D005910:Glioma; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011759:Pyrrolidines / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013134:Spiperone / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001549:Benzamides; D001552:Benzazepines; D017931:DNA Primers; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D011759:Pyrrolidines; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; C063525:epidepride; D013134:Spiperone; D000262:Adenylyl Cyclases; D004298:Dopamine