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

Abstract

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) 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 FZD5 for signal specification. Similar data were obtained for FZD4 and FZD10 suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD5 mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD5 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.

SAG1.3-derived Frizzled-targeting negative allosteric modulators

“…One of them is the signalosome model, according to which the initiation of WNT signaling is achieved by WNTs serving as crosslinkers between FZDs and certain co-receptors, e.g., LRP5/6 for WNT/β-catenin signaling thereby specifying the signaling outcome 38,39 . While the signalosome model excludes intrinsic receptor dynamics [40][41][42] , FZDs behave -in agreement with what is known for other GPCRs -as dynamic entities, sometimes referred to as molecular machines 24,43,44 . The latter model includes an allosteric coupling between the CRD -the orthosteric WNT binding site -and the intracellular transducer coupling interface 23,24,45,46 .…”

Structural Basis of Frizzled 7 Activation and Allosteric Regulation

“…In that regard FZDs differ from SMO, which lacks the proline and accommodates G protein association through parallel outward shift of TM6 instead of a kink. While it is now well established that FZDs are binding and activating heterotrimeric G proteins (2, 3), systematic analysis of FZD mutants have unraveled that they tend to prefer DVL over G protein coupling suggesting that distinct conformational substates de ne transducer selectivity (33). Interestingly, mutations affecting the molecular switch of FZD 6 , speci cally R 6.…”

“…C, D) permitting only a restricted TM6 opening (11°, 5.5Å). Hence, the open conformation of FZDs remains suboptimal for G protein coupling providing a rational explanation for the limited capacity of FZDs to couple to heterotrimeric G proteins and their propensity to display selectivity towards DVL over heterotrimeric G proteins (33). Interestingly, R/K 6.32 is frequently mutated in cancers with obvious consequences on TM6 dynamics, eventually impacting receptor signaling pro les.…”