Abstract

To refine further the structure-activity relationships of D₁ dopamine receptor agonists, we investigated the roles of three conserved serine residues [Ser198(5.42), Ser199(5.43), and Ser202(5.46)] in agonist binding and receptor activation. These transmembrane domain 5 (TM5) residues are believed to engage catechol ligands through polar interactions. We stably expressed wild-type or mutant (S198A, S199A, and S202A) D₁ receptors in human embryonic kidney cells. These receptors were expressed at similar levels (approximately 2000 fmol/mg) and bound the radioligand [³H]R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), although S198A and S199A displayed significant losses of affinity compared with that for wild-type receptors. The endogenous agonist, dopamine, had losses of potency at each of the mutant receptors. We tested cyclohexyl-substituted isochroman, carbocyclic, and chroman bicyclic dopamine analogs and found that the mutations affected the chroman to a lesser extent than the other compounds. These results support our hypothesis that the decreased D₁ activity of chroman analogs results from a ligand intramolecular hydrogen bond that impairs the ability of the catechol to engage the receptor. Sensitivities of these rigid catechol agonists to the effects of the serine mutations were dependent on ligand geometry, particularly with respect to the rotameric conformation of the ethylamine side chain and the distance between the amino group and each catechol hydroxyl. Functional experiments in striatal tissue suggest that the ability to engage TM5 serines is largely correlated with agonist efficacy for cAMP stimulation. These results provide a new understanding of the complexities of D₁ ligand recognition and agonist activation and have implications for the design of rigid catechol ligands.