Human dopamine signaling pathway induces and facilitates dopamine neurotransmission through the mesolimbic dopaminergic pathway, which modifies reward-related behaviors and is associated with the development of many diseases, including schizophrenia, Huntington's disease, cognitive disorders and Parkinson?s disease. Mounting evidence suggests that this important signaling pathway is constructively regulated by the dopamine receptors (DRs). Thus, making members of this membrane protein family highly promising therapeutic targets as supported by both pre-clinical and clinical studies. Although some agonists of receptors in the human DR (hDR) family members (e.g. for dopamine D1-like receptors) are being intensively studied for therapeutic intervention, their success has been greatly hampered due to poor adherence and efficacy, or due to associated side effects. Relatedly, the polypharmacology of dopamine D1-like receptor and other hDRs have been discovered in recent studies. New knowledge based on structures of D1-like receptors (D1R and D5R) will not only reveal their signaling mechanisms, but also provide new understanding that can be exploited to facilitate rational drug design. hDRs belong to the G protein-coupled receptor (GPCR) family which is notorious for difficulties in generating diffraction-quality crystals that are essential for the determination of high-resolution structures by X-ray crystallography. This R&D proposal aims to develop a comprehensive and robust platform for structural and functional studies of dopamine D1-like receptors and complexes of hDRs with G proteins, for screening high- affinity nanobodies, antibodies, and ligands targeting these receptors. This platform will involve multiple steps that are closely interconnected and looped through a forward and backward feedback system. The PI has now also established strong collaborations with other research groups with different expertise as alternative approaches, including X-ray free electron laser, Microcrystal Electron Diffraction technique, etc.
Three specific aims are proposed: (1) Structure/function studies of human D1R (hD1R) in the inactive state, (2) Using X-ray crystallography and computational biology approaches to study the putative active state of hD1R, and (3) Establishing optimized approaches for determining the structure of complexes of hD1R with G protein partners using EM imaging. The significance of this study is multi-fold on dopamine signaling pathway and related drug discovery studies: 1) we will gain insights into dopamine D1-like receptor functionalities and allosteric modulations, 2) we will be able to screen extensively to identify new high-affinity ligands for hDRs, 3) characterize the mechanisms of DR signaling and ligand selection between different dopamine receptor subfamilies, 4) stimulate hDR structure-based drug design, 5) examine hDRs/G protein complex signaling and reveal the activation mechanism, and 6) pave the road for the application of cryoEM technology on difficult membrane protein targets in the future.
Dopamine receptors play important roles in human dopamine signaling pathway; therefore, targeting these membrane proteins provide promising therapies for many diseases, including schizophrenia, Huntington's disease, cognitive disorders and Parkinson?s disease. Here we propose to resolve the structure of human dopamine D1 like receptor (D1R), along with the relevant G protein complexes. We expect the structural information we obtain from this study will facilitate allosteric modulation and activation mechanism studies, and therefore benefit rational design of safer and more efficacious therapeutics.
