Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD 1-10 ) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive. Here we employ a structure-driven mutagenesis approach in combination with an extensive panel of functional signaling readouts to investigate the importance of conserved state-stabilizing residues in FZD 5 for signal specification. Similar data were obtained for FZD 4 and FZD 10 suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD 5 mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD 5 and FZDs in general prefer coupling to DVL rather than heterotrimeric G proteins and that distinct active state micro-switches in the receptor are essential for pathway selection arguing for conformational changes in the receptor protein defining transducer selectivity.