A disorder-to-order structural transition in the COOH-tail of Fz4 determines misfolding of the L501fsX533-Fz4 mutant

Abstract

Frizzled 4 belongs to the superfamily of G protein coupled receptors. The unstructured cytosolic tail of the receptor is essential for its activity. The mutation L501fsX533 in the fz4 gene results in a new COOH-tail of the receptor and causes a form of Familial exudative vitreoretinopathy. Here we show that the mutated tail is structured. Two amphipathic helices, displaying affinity for membranes and resembling the structure of Influenza Hemagglutinin fusion peptide, constitute the new fold. This tail induces the aggregation of the receptor in the Endoplasmic Reticulum and it is sufficient to block the export to the Golgi of a chimeric VSVG protein containing the mutated tail. Affecting the tail's structure, net charge or amphipathicity relocates the mutated Fz4 receptor to the Plasma Membrane. Such disorder-to-order structural transition was never described in GPCRs and opens a new scenario on the possible effect of mutations on unstructured regions of proteins.

Pharmacological folding chaperones act as allosteric ligands of Frizzled4

“…As previously shown 26,27 , HA-Fz4-WT was localized in the Golgi complex and at the PM, whereas HA-Fz4-FEVR appeared to be trapped intracellularly, mainly in the ER ( Fig. 1b Tables 4 and 5).…”

“…This difference most likely indicates that the ligands support HA-Fz4-FEVR molecules in the process of folding rather than rescuing those that are already aggregated. The latter accumulate in the HA-Fz4-FEVR stable HEK293 cell line before treatment with compound 26,27 , and, because they have a covalent nature 27 , they could indeed be difficult to rescue.…”

“…The mutated C-terminal tail of Fz4-FEVR improperly interacts with the ER lipid bilayer, disturbing receptor stability and inducing its aggregation and retention in the ER (Fig. 1a) 27 . Notwithstanding, Fz4-FEVR localization at the PM can be rescued by overexpressing the protein chaperone α-B crystallin 26 .…”

“…In untreated cells, Fz4-FEVR forms covalent aggregates 27 . As shown by western blot analysis of lysates of Fz-FEVR-expressing cells, FzM1 treatment increased Fz4-FEVR solubility in a dose-response manner ( Supplementary Fig.…”

“…In humans, despite being rare, they lead to the development of familial exudative vitreoretinopathy (FEVR), a pathology resulting in aberrant vascularization of the retina during embryo development 25 . Among the mutations, the frameshift L501fsX533 of Fz4 shows autosomal dominant inheritance and causes a conformational defect by generating a different and shorter C-terminal cytosolic tail that hampers signaling of the mutant receptor (henceforth referred as Fz4-FEVR) and its correct folding and transport to the cell plasma membrane (PM) [25][26][27] .…”