Differential Activation of Adenylate Cyclase and Receptor Internalization by Novel Dopamine D1 Receptor Agonists

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Abstract
Structurally dissimilar dopamine D1 receptor agonists were compared with dopamine in their ability to activate adenylate cyclase and to internalize hemagglutinin-tagged human D1 receptors in a stably transfected human embryonic kidney cell line. Thirteen dopamine D1 receptor agonists were selected rationally from three different structural classes: rigid fused ring compounds [dihydrexidine, dinapsoline, dinoxyline, apomorphine, and (5aR,11bS)-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-azacyclopent-1-ena[c]-phenanthrene-9,10-diol (A86929)]; isochromans [(1R,3S)-3-(1′adamantyl)-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A77636) and (1R,3S)-3-phenyl-1-aminomethyl-3,4-dihydo-5,6-dihydroxy-1H-2-benzopyran (A68930)]; and benzazepines [7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF38393), (±)-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF77434), 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), 3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H-3-benzazepine (SKF83959), R(+)-6-chloro-7,8,-dihydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82957), and R(+)-6-chloro-7,8,-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297)]. The working hypothesis was that some agonists have differential effects on adenylate cyclase versus receptor internalization that could be correlated to the structural class of the agonist. First, the affinity for the hemagglutinin-hD1 receptor and the intrinsic activity and potency of adenylate cyclase activation were determined for each compound. The internalization time course and internalization efficacy were then determined for each agonist. It was surprising that internalization efficacy was found to be independent of either agonist structural class or affinity. Only agonists that had both high adenylate cyclase functional potency and high intrinsic activity caused internalization. In addition, four agonists from two structural classes were identified that were capable of fully activating adenylate cyclase without eliciting an internalization response. This study provides the first extensive characterization of D1 receptor internalization in response to structurally diverse agonists and, at least for the D1 receptor, shows that functional selectivity is not predictable by simple structural examination. These data are consistent with the hypothesis that functional selectivity reflects subtle ligand-induced conformational changes as opposed to simple agonist trafficking among discrete receptor active states.

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