Dopamine receptor structure/function studies.

Abstract

The general aim of this dissertation was to investigate potential mechanisms linking ligand binding and signal transduction in seven transmembrane helix, G protein-coupled receptors. For this purpose, dopamine D$\sb1$ and D$\sb2$ receptors were analyzed at the ligand binding pocket, the third cytosolic loop, and transmembrane-embedded kinks which potentially link ligand binding and G protein-coupling. The first study investigated variability in the D$\sb2$ third cytosolic loop, a region putatively involved in G protein-coupling. The results indicate that, due to this variability, the long and short isoforms of D$\sb2$ exhibit differences in binding pocket geometries which are especially well detected by substituted benzamide antagonists and are observed only in particular expression conditions. The third intracellular loop variability may be responsible for differential coupling of the D$\sb2$ isoforms to some intracellular or membrane-bound factor(s) other than G proteins. In the second study, conserved hydrophobic residues in D$\sb2$, predicted to be important for stabilization of ligand binding or for propagation of agonist-induced conformational changes, were investigated by mutagenesis. Two phenylalanines located on the fifth helix were found to be critical for agonist and antagonist binding. One seventh helix phenylalanine and one sixth helix leucine appear to be critical for linking ligand binding and signal transduction since their mutations led to dissociation of these receptor functions. The third project investigated the potential role of proline-adjacent residues in D$\sb1$ receptor structure and dynamics. Mutation of residues in the fifth and sixth helices indicated that the nature, or volume, of the residue immediately adjacent to proline on the N-adjacent side can alter both ligand binding and signal transduction. One of these mutations, an alanine substitution of the sixth helix leucine286, conferred partial constitutive activity to the D$\sb1$ receptor, characterized by elevated basal cAMP levels and increased agonist potencies. This is the first report of a constitutively active dopamine receptor mutant. Together, these results have expanded the study of structure/function relationships in dopamine receptors by identifying several residues in the D$\sb1$ and D$\sb2$ receptor subtypes which appear to be important for ligand binding, signal transduction, and perhaps for conformational changes linking these two receptor functions.