Molecular modelling studies on Frizzled receptors
Abstract
The superfamily of G protein-coupled receptors (GPCRs) mediates effects of many endogenous and exogenous substances such as small molecules, peptides, lipids, ions, and odorants. According to homology, GPCRs were grouped into Classes A, B, C, F (Frizzleds), adhesion receptors, and other 7 transmembrane (TM) spanning receptors. Frizzleds (FZDs) regulate a number of processes during embryonic development, stem cell regulation, and adult tissue homeostasis.
Deregulation of FZDs leads to pathogenesis, including, but not limited to, cancer and neurologic disorders; thus, making them attractive drug targets. In mammals, there are 10 Frizzleds (FZD1–10), which are activated by the WNT family of lipoglycoproteins through interaction with the extracellular cysteine-rich domain (CRD) of FZD. Our group has shown that FZD6 dynamically dimerizes in an agonist-dependent fashion and that the dimer interface of FZD6 is formed by the transmembrane α-helices four and five. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of signalling.
We plan to expand our understanding of molecular mechanisms of FZD activation and signal initiation by analysis of membrane localization, dimerization status and constitutive activity of mutants of FZD6. Mutation of R416 enhances the receptors ability to negatively impact on the WNT/b-catenin pathway, prevents dimerization and increases receptor internalization and turnover. R416 is located at the lower end of TM6 mediating interaction with TM7 thereby providing a potential lock mechanism that could explain the mutations apparent constitutive activity. Furthermore, R416 is conserved in all human FZDs except for FZD4 and FZD9. This mutation has also been found in many forms of cancer, including uterine and bladder. In addition to that we have started introducing unnatural amino acids TM4 and TM5 – the site of dimer interface. This will enable us to further map the dimer interface in greater detail to understand molecular rearrangements in the receptor complex upon ligand-induced activation. A similar approach of orthogonal labelling and photocrosslinking is currently used to map and define WNT-FZD interaction sites on the extracellular CRD and also on the extracellular surface of the receptor molecule
Our goal is now to validate and support our models based on currently available mutants and to predict novel sites for mutagenesis experiments in the dimer interface using MD simulations of the receptor dimer embedded in a POPC lipid bilayer and solvated in water. A high resolution crystal structure of FZD6 is so far not available and we are therefore using a FZD6 model based on crystal structures of Smoothened, a closely related Class F receptor. In continuation we plan to complement receptor function analysis, mutagenesis and biochemical approaches with computational modelling and MD simulations to fully understand structural features and dynamics ranging from WNT-FZD binding, conformational changes in the receptor and effector interaction on the intracellular side. A better understanding of agonist-induced FZD activation on detailed molecular level will aid in our attempts to target these receptors pharmacologically aiming for improvements in therapy of diverse forms of cancer and fibrosis.
