Abstract
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.
Highlights
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD110) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families
We propose that SMO activation relies on cholesterol binding to the 7TM core of the receptor maintaining a straight transmembrane helix 6 (TM6), whereas activation-associated conformational changes of FZD6 rather manifest in a kinked TM6 and are independent of cholesterol binding in the 7TM core of the receptor
In order to broadly compare all class F GPCRs, we performed a sequence alignment of class F receptors in human, orangutan, rat, mouse, dog, chicken, frog, and fruit fly based on the sequences obtained from the UniProtKB/Swiss-Prot database (Supplementary Data 1)
Summary
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD110) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. Studying GPCR activation has led to the common agreement that an outward movement of the transmembrane helix 6 (TM6) is the main hallmark of the active GPCR conformation[7,8] This reorganization of the TM6 involves a complex interaction network within the receptor structures and is tied to the presence of the conserved proline residues P5.50, P6.50, and P7.50 allowing helix dynamics Our recent work on class F structure-function aspects emphasized that two distinct, conformational activation pathways exist in FZDs. On the one hand, interaction with G proteins is accompanied by the opening of a molecular switch (R6.32/W7.55) in the receptor core between. The differences are most distinct when comparing ligand binding and downstream signaling along the FZD/β-catenin or the SMO/GLI pathways[21,22]
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