D1 AND D2 DOPAMINE RECEPTOR BIASED AND HETEROMERIC SIGNALING: IMPLICATIONS FOR PROTEIN CONFORMATIONAL FLUIDITY

Abstract

The D1R and D2R dopamine receptor (DAR) subtypes represent the two most abundant and highly targeted DARs, but the precise mechanisms of drug action at the molecular level are not well understood. A revolution in the understanding of G protein-coupled receptors depicts receptors as dynamic, conformationally fluid proteins. This led to the idea that receptor heteromerization or ligand binding can promote functionally selective/biased signaling. In this vein, two aspects of DAR signaling are studied: D1R-D2R heteromerization and biased D2R signaling. In Chapter 2, the pharmacology and signaling mechanism for the D1R-D2R heteromer is investigated. Diverging from canonical D1R-Gs/olf protein and D2R-Gi/o protein coupling, the D1R-D2R heteromer was proposed to couple to Gq protein-mediated Ca2+ mobilization. In Chapter 2, it is shown that D1R-D2R-mediated Ca2+ signaling may not be completely Gq protein-dependent or heteromer-specific, and may largely depend on Gi/o protein, Gs/olf protein, and Gβγ signaling. Furthermore, SKF83959 (previously reported as a D1R-D2R selective agonist) has significant cross-reactivity to other receptors, warranting careful interpretation of its use in vivo. In Chapter 3, a high-throughput screen was conducted to interrogate a small-molecule library for novel D2R agonists using a Ca2+ mobilization assay. Following additional orthogonal screening of cAMP modulation and β-arrestin-2 recruitment, a G protein biased D2R agonist was identified (MLS1547). MLS1547 analogs were tested using the cAMP accumulation and β-arrestin-2 recruitment assays. These results provided the basis for pharmacophore modeling and molecular docking analyses to build structure-activity relationships (SAR) of the functionally selective properties of this series of compounds. Chapter 4 extends the pharmacological profiling of MLS1547 by showing that MLS1547 stimulates a low but significant degree of D2R internalization. The pharmacophore model is also confirmed and extended by testing additional MLS1547 analogs and developing a refined SAR. In Chapter 5, another high-throughput screen was conducted to detect β-arrestin-2 biased D2R selective ligands. Although one β-arrestin-2 biased agonist was identified, further testing was not possible, as a source for the compound was not available. Taken together, these data expand the understanding of the spectrum of signaling capabilities available to the D1R and D2R by investigating two novel mechanisms of signaling.