Ligand Specificity and Activation of Receptor Guanylyl Cyclases - Abstract Receptor guanylyl cyclases (rGCs) catalyze the conversion of GTP to cyclic GMP (cGMP), a key second messenger that regulates diverse physiological processes across multiple human tissues. Depending on their subtype and tissue distribution, rGCs are implicated in a wide range of diseases, including hypertension, skeletal dysplasia (dwarfism), secretory diarrhea, and retinal degeneration. Despite decades of research establishing their physiological importance, therapeutic strategies targeting rGCs remain limited, largely due to a lack of detailed structural and mechanistic understanding of these receptors. rGCs present several intrinsic challenges for structural and functional characterization: they are single-pass transmembrane receptors with tissue-specific expression, low endogenous abundance, and significant conformational flexibility. These properties have historically impeded high-resolution structural analysis and hindered drug discovery efforts. The long-term goal of my research program is to overcome these challenges and elucidate the molecular mechanisms governing rGC activation under both physiological and pathological conditions. Among the five known human rGCs, GC-A, GC-B, and GC-C are activated by their cognate extracellular peptide ligands, while GC-E and GC-F are regulated by intracellular signaling cues. Recently, we successfully determined the first full-length structures of GC-A using single-particle cryo-electron microscopy (cryo-EM), revealing its activation mechanism and resolving ligand interactions at atomic resolution. These findings establish a powerful framework for further investigation. In this proposed research program, we will extend our structural and mechanistic studies to GC-B and GC-C. GC-B, activated by the cyclic peptide C-type natriuretic peptide (CNP), is a promising therapeutic target for skeletal growth disorders. GC-C, exclusively expressed in the gastrointestinal tract, is activated by intestinal cyclic peptides guanylin and uroguanylin, and also serves as the receptor for the enterotoxigenic bacterial toxin STa. To date, no active-state structures of GC-B or GC-C have been reported, and their ligand-binding interfaces remain poorly defined. We will leverage the powerful, bespoke framework developed for GC-A to overcome the longstanding barriers to understanding GC-B and GC-C, providing the first atomic-level blueprints of their activation mechanisms. The proposed comprehensive study aligns closely with the core mission of NIGMS by advancing our understanding of the fundamental principles and mechanisms of receptor signaling, while also laying the groundwork for future strategies in the treatment of diseases targeting rGCs.
