Abstract or Keywords
In this study, we employed site‐directed mutagenesis to explore the complex hydrogen bonding interactions that are critical for D1 dopamine receptor activation. We stably expressed wild‐type and three transmembrane 5 serine to alanine mutants (S198(5.42)A, S199(5.43)A, and S202(5.46)A) in HEK 293 cells, and evaluated binding affinity and potency of cAMP production for many structurally diverse ligands. Each of these cell lines expressed similar levels of receptor (approximately 2,000 fmol/mg), and bound [3H]SCH‐23390. The radioligand exhibited significant losses of affinity, however, at S198A and S199A. Dopamine, tetracyclics (e.g. dihydrexidine) and cyclohexyl‐substituted bicyclics similarly suffered losses of affinity and potency at the mutant receptors, where S202A produced the largest disruption of binding and activity. Interestingly, the phenylbenzazepines (e.g. SKF‐38393) demonstrated reduced affinity and potency for each mutant, but were least affected by S202A. Uniquely, the non‐catechol ergolines (e.g. CY 208–243) were more potent at S198A, whereas S199A and S202A produced relatively minor losses of affinity and potency. These results demonstrate that these structurally diverse ligands uniquely interact with the D1 binding pocket, and present a new understanding of the complexities of D1‐ligand recognition and activation. This work was supported by NIH grants MH42705 and MH60397.