INVESTIGATING THE DIMERIZATION OF FRIZZLED 6 (FZD₆) RECEPTOR

Frizzled (FZD) receptors are involved in cellular processes such as proliferation and migration. The FZD class, a separate family of G protein-coupled receptors (GPCR), has 10 mammalian isoforms, all contain 7 Trans Membrane domains (TM). The extracellular region in all FZDs contains a highly conserved cysteine - rich domain, the Frizzled domain, which interacts with its ligands. During their function, FZDs go through dynamic dimerization, as revealed by dcFRAP and live cell imaging experiments. A model of FZD₆ (isoform 6) was designed based on the crystal structure of one of its class members, the similar smoothened (SMO) protein. The model implies a possible dimerization through the interaction between two of its 7 trans-membrane (TM) domains, TM4 and TM5, from two different monomers. In this study we aim to quantify and characterize this dimerization process through the interaction between these two domains of FZD₆.

To characterize this interaction we synthesized and purified peptides derived from these two TM regions. We used Circular Dichroism (CD) spectroscopy to determine the proper conditions in which the TM regions maintain their native α-helical structure and found that in 0.5% DDM both peptides are helical.

To quantify the interaction between the two peptides, Isothermal Titration Calorimetry (ITC) experiments were performed. Using this method we determined that the dissociation constant (k_d) is in the micro molar range, which indicates a moderate binding affinity. In addition to WT TM5 peptide we examined mutated peptides which are predicted to impair the dimer and as an outcome, interfere with FZD₆ function. CD analysis shows that asides from one, the mutated peptides gain an alpha helical structure in lower DDM concentrations than the WT peptide.