The G protein-coupled receptors (GPCRs) mediate effects of many endogenous and exogenous substances such as small molecules, peptides, lipids, ions and odorants. According to sequence homology, GPCRs are 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.
In mammals, there are 10 FZDs (FZD1–10), which are activated by the WNT family of lipoglycoproteins likely through interaction with the extracellular cysteine-rich domain (CRD) of FZDs. At the cytosolic side FZDs interact with Dishevelled (Dvl) and G proteins. Deregulation of FZDs leads to pathogenesis, including cancer and neurological disorders thus making them attractive drug targets. In my studies I am planning to undertake molecular modelling and biochemical approaches to study activation mechanisms of FZD6. A crystal structure of FZD6 is not available and we are therefore using a FZD6 model based on crystal structure of Smoothened, a closely related Class F receptor.
We plan to expand our understanding of molecular mechanisms of FZD activation and signal initiation by analysis of membrane localization, ligand sensitivity and constitutive activity of different mutants of FZD6. Until now we have discovered that mutation of R416 enhances the receptor’s ability to negatively impact on the b-catenin pathway and more importantly increases receptor ligand sensitivity. (Wright and Kozielewicz, 2018, submitted; used SNIC resources) R416 is located at the lower end of the TM6 mediating interaction with W493 of the TM7 thereby providing a potential polar lock mechanism that could explain the mutant's apparent constitutive activity. Similar interactions have been reported for class B GPCRs. (Wootten, Biochemical Pharmacology, 2016) We are currently analysing polar and non-polar interactions in receptor structures aiming to pinpoint general mechanisms of receptor activation in Class B and F family GPCRs. Furthermore, R416 (or a corresponding basic lysine) is conserved in all human FZDs. This mutation has also been found in many forms of cancer, including uterine and bladder cancer. In addition to that, using molecular modelling studies, we identified other relevant residues in other TMs which potentially also contribute to switching between active/inactive receptor conformations.
Another aim of our project is to decipher the G protein binding site of the FZDs. In contrast to the FZD–Dvl binding site (Tauriello, PNAS, 2012), the G protein interface has not been mapped, which is partially due to the lack structural information e.g. from an active conformation crystal structure of a class F GPCR. Structural alignments and molecular dynamics simulations will be used to predict potential sites which can be subsequently mutated to investigate FZD–G protein interface biochemically. Receptor activation and G protein coupling will assessed in biochemical studies using approaches like: Bioluminescence resonance energy transfer (BRET) and pErk AlphaScreen. In addition to that, we will employ mini G proteins (Wan, JBC, 2018), which serve as conformational sensors of the active receptor state directly probing a conformational switch from active to inactive receptor.