Abstract
AbstractReceptors of the Frizzled family initiate Wnt ligand-dependent signaling controllingmultiple steps in organism development and highly conserved in evolution.Misactivation of the Wnt/Frizzled signaling is cancerogenic. Frizzled receptorslaunch several signaling cascades: the canonical pathway regulating beta-catenin-dependent transcription; the planar cell polarity pathway polarizing thecytoskeleton within the epithelial plane; and the calcium pathway. Frizzledreceptors possess seven transmembrane domains and their signaling depends ontrimeric G proteins in various organisms. However, Frizzleds constitute adistinct group within the G protein-coupled receptors (GPCR) superfamily, andFrizzled signaling can be G protein-independent in some experimental setups, which led to concerns about the GPCR nature of Frizzled. Here we demonstratethat human Frizzled receptors can directly bind the trimeric Go protein in apertussis toxin-sensitive manner. Furthermore, addition of Wnt ligands elicitsFrizzled-dependent guanine nucleotide exchange on Go. An excess of secretedFrizzled-related protein (a Wnt antagonist) prevents Go activation, as doespretreatment of Go with pertussis toxin. These experiments provide a biochemicalproof of the GPCR activities of Frizzled receptors and establish an in vitro assay tomonitor Frizzled activation by Wnt ligands, applicable for the high-throughputagonist/antagonist screening.
Highlights
G protein-coupled receptors (GPCRs) physically bind to trimeric G proteins. In their agonist-activated form, GPCRs catalyze the substitution of GDP for GTP on the G-alpha subunit of trimeric G proteins - an event causing dissociation of the trimeric complex into the G -GTP and the -heterodimer [12]
To prove the capacity of Fz to directly interact with trimeric G proteins, we expressed the human Fz-1 receptor in E.coli as a recombinant plasma membranedirected protein tagged with MBP
We find that pretreatment of Go with pertussis toxin (Ptx), but not the control treatment, abrogated the ability of this G protein to be activated by Wnt3a-human Fz-1 receptor (hFz1) in the in vitro assay (Figure 2B)
Summary
For a formal biochemical proof of the GPCR nature of Frizzled (Fz) receptors, their capacity to physically and directly bind the trimeric G proteins must be demonstrated, as well as the ability of the Wnt ligands to stimulate Fz-dependent guanine nucleotide exchange on these G proteins. To prove the capacity of Fz to directly interact with trimeric G proteins, we expressed the human Fz-1 receptor (hFz1) in E.coli as a recombinant plasma membranedirected protein tagged with MBP (maltose-binding protein, Figure 1A).
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