A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection

Abstract

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

Wnt11/Fzd7 signaling compartmentalizes AKAP2/PKA to regulate L-type Ca2+channel

“…The unique sequence and structural features of the C-terminal tail of Frizzleds are noted to hinder the binding of traditional GPCR ligands 24,26 . Nevertheless, increasing evidence are indicative of their ability to signal through heterotrimeric G proteins 25,27,45 . Similarly, Wnt ligands are able to regulate cAMP levels and thus activate the cAMP/PKA pathway in diverse contexts [46][47][48] .…”

“…Wnt ligands act through several receptors and co-receptors, most notably via the Fzd family of receptors belonging to Class F GPCRs 24,25 . While structurally different from Class A receptors including β-AR 24,26 , the concept that Fzds do signal through heterotrimeric G proteins gained traction in recent years 25,[27][28][29][30][31] . The identification of the signaling components downstream of Fzd-G protein coupling, however, remains unresolved.…”

Biochemical mechanisms of vertebrate hedgehog signaling

“…3F). This ionic lock is broken by known oncogenic mutations that activate SMO and is also conserved in the FZD group of WNT receptors (Wright et al, 2019). SMO activation may result in the rupture of this ionic lock, leading to the outward movement of TM6 and the consequent exposure of a new molecular surface to engage a cytoplasmic effector.…”

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

“…University, Georgia, Canada (Wright et al, 2019). The mouse SMO sequence was subcloned into an empty N-terminally tagged Nluc vector containing 5-HT3A signal peptide using BamHI and XbaI restriction sites.…”