Structural and Mechanistic Bases for Ligand Specificity of α1-Adrenergic Receptors

PROJECT SUMMARY/ABSTRACT α1-adrenergic receptors (ARs) are G-protein-coupled receptors (GPCRs) that play a critical role in regulating various physiological functions and are implicated in pathological conditions, including heart failure and Alzheimer’s disease. However, the precise roles of the three α1-AR subtypes, including α1A, α1B, and α1D, remain poorly understood, largely due to the limited availability of subtype-selective ligands that enable targeted activation for individual study. Our research program aims to address these significant gaps by providing structural and mechanistic insights into the ligand specificity of α1-ARs through the use of cryo- electron microscopy (cryo-EM), combined with biochemical and cellular studies. The long-term goal is to develop highly selective ligands that exhibit subtype specificity, thereby facilitating a comprehensive understanding of the distinct physiological roles of each subtype and supporting the development of novel therapeutics targeting each subtype. Our research will focus on two categories of ligands: agonists and positive allosteric modulators (PAMs). Agonists bind to the orthosteric binding site of the receptor to activate it, while PAMs bind to an alternative site to enhance the affinity and efficacy of agonists. Currently, there are only three α1A-AR selective agonists and one α1A-AR selective PAM available for the entire α1-AR family, with no selective agonists or PAMs identified for α1B-AR or α1D-AR. A major challenge in developing subtype-selective ligands is the absence of active state structures of α1-ARs. The first active state structure of α1A-AR was determined by my postdoctoral work in 2023, alongside similar efforts from another group. To date, no active state structures of the α1B or α1D subtypes have been reported. My independent research program at Binghamton University will leverage cryo-EM to generate structural frameworks for understanding the ligand specificity of α1-ARs. We will determine cryo-EM structures of the three α1-AR subtypes bound to non-selective endogenous agonists, as well as selective agonists and PAMs. Through systematic structural comparisons, we aim to identify key residues and ligand functional groups that are critical for subtype specificity. Additionally, we will validate the roles of these identified residues through mutational and functional studies. This research program builds upon my extensive experience with GPCR structural and mechanistic studies, as evidenced by my recent publications and preliminary data described in the Research Strategy. My research group has established the necessary knowledge, techniques, and tools to successfully execute our research program. Our findings will pave the way for the structure-based design of novel subtype-selective agonists and PAMs and contribute to addressing key gaps in understanding the physiological and pathological roles of individual α1-AR subtypes.