Abstract
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.
Highlights
From the $Veterans Affairs Medical Center and Departments of Pharmacology, Psychiatry, and §Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, the §Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, and the Wepartment of Genetics, Harvard Medical School an2 McLean Hospital, Belmont, Massachusetts 02178
The chimeric receptors had affinities for three D1-selective ligands and the D2-selective agonist, quinpirole, that were intermediate between D l and D2 receptor affinities for the drugs
TheD l sequence fromthe amino terminus to the carboxyl-terminal enodf the second extracellular region was amplified using a T7 polymerase promoter 23-mer and a 36-mer that is the reversecomplement of 5 ' - m structed in such a way that the expressed chimeras had D l receptor sequence at the amino-terminal end anDd2 sequence at the carboxyl-terminal end.All of the chimeras andwild-type D l (13) and D2 (14)receptorswere stably expressed in C, glioma cells, and the binding of three radioligandswas assessed
Summary
Research Service (151LL), VA Medical Center, 3710 SW US Veterans HospitalRd., Portland, OR 97201. The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; Gi, G protein that inhibits adenylyl cyclase; Gs, G protein that stimulates adenylyl cyclase; TM, transmembrane(s);chloroAPB, ~~~-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-lH-3benzazepine. CH4 structure, affinities for selective agonists and antagonists, and r transduction mechanisms. Like aZ-and &adrenergic receptors, D l and D2 receptors are similar in some respects, with about 25% amino acid sequence identity, primarily in the putative TM regions, and similar affinities for some ligands. The qualitatdivifeferences in second messenger coupling and the many quantitativdeifferences in ligand binding indicated that analysis of DUD2 receptor chimeras would.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
