Site-directed mutagenesis of Tyr417 in the rat D2 dopamine receptor.

The D2 dopamine receptor is a member of the family of Gprotein linked receptors. They are thought to consist of seven hydrophobic transmembrane domains (TMD) linked together by intraand extra-cellular loops. Within this group of receptors there is a high degree of homology, particularly within the membrane spanning regions [ 11, and within these domains certain conserved amino acids have been implicated as possible sites for receptor-ligand interaction. Evidence for their involvement comes from mutagenesis studies on other members of this group, including studies on the fi adrenergic receptor [2-51 and more recently the D2 receptor [6,7], and also from pH and chemical modification experiments [8,9]. In this study Tyr417 in TMD VII has been targeted, primarily as a result of chemical modification [lo] and computer modelling studies [ 111, as a possible site for receptor-ligand interaction. Tyr417 in TMD VII of the rat Dqlong) dopamine receptor has been mutated to Phe417 by a single base-pair substitution: TACTTC. This has been achieved by performing site-directed mutagenesis on the rat D2(long) cDNA cloned into pBluescript KS+ (provided by Phillipe Vernier). The mutated cDNA was then ' subcloned into D2pSVL, a mammalian expression vector containing the rat D2(lon cDNA. The subcloning was performed so that only T d s VI and VII from the mutated cDNA were exchanged with those of the native D2pSVL vector. The fidelity of the subcloning was confirmed by restriction enzyme digest analysis, and dideoxy sequencing. In order to characterise the effect of the Phe417 mutation, the mutant and native constructs were transfected into COS-7 cells using the DEAE-Dextran method of transfection, and expressed transiently. Membranes prepared from the transfected cells were then used to perform competition experiments with [3H]spiperone to determine the effect of the Phe417 mutation on the affinity of the receptor for a range of classical antagonist and substituted benzamide drugs. For these experiments, between 301OOpg of membrane protein was incubated with 0.25nM [3H]spiperone, competing drug, and buffer (20mM HEPES pH7.4, ImM EDTA, 1mM EGTA, (120nM NaCl only added for the binding of substituted benzamide drugs)) [12] to a final volume of lml. Saturation analysis was performed using a range of [3H]spiperone concentrations from 4M-15pM. Specific binding was determined as the binding inhibited by 3pM (+)butaclamol for the native receptor, and 10pM for the Phe417 mutant (see below). The reactions were incubated for 45 minutes at 25OC before harvesting on a Brandel cell harvester. The filters were washed with three 4ml washes of phosphate buffered saline (0.14M NaCI, 3mM KCI, 1.5mM KH2PO4 and 5mM Na2HPO4, pH7.4) at 4OC. The filters were then soaked in 2ml of Optiphase Hisafe 3 scintillation fluid for 6 hours, and then the bound radioactivity determined by liquid scintillation counting. Transient expression of the native and Phe417 mutant cDNAs resulted in expression of the receptors at approximately 5OOfmoVmg membrane protein. A difference in the affinity of the mutant receptor for [3H]-spiperone compared with the native receptor was seen as determined by saturation analysis, and the value was used in the analysis of competition data. A similar effect was seen with (+)-butaclamol with the mutant receptor, so 10pM (+)-butaclamol was used to define non-specific binding in Classical native 4.21k0.59 domperidone 1.67M.10 5.57M.85 cis-flupentixol 7.59k1.12 31.9k5.05 haloperidol 2.37M.85 7.99k0.49 3.4 spiperone I 0.053M.01 I 0.31k0.04 I 5.8 Substituted Benzamide