Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6

Abstract

Frizzleds (FZDs) are classified as G-protein-coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD6 regulates convergent extension movements, and its C-terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD6, Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD6 precouples to Gαi1 and Gαq but not to GαoA,Gαs, and Gα12 proteins. G-protein coupling is measured as a 10-20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross-linking. The FZD6 Arg511Cys mutation is incapable of G-protein precoupling, even though it still binds DVL. Using both FRAP and Förster resonance energy transfer (FRET) technology, we showed that the FZD6-Gαi1 and FZD-Gαq complexes dissociate on WNT-5A stimulation. Most important, G-protein precoupling of FZD6 and WNT-5A-induced signaling to extracellular signal-regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD6-G-protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G-protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G-protein, and DVL function.

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13

“…FZD 6 exists in an inactive-state complex with heterotrimeric Ga i and Ga q (Kilander et al, 2014b) similar to what was observed for the muscarinic M 3 receptor (Qin et al, 2011). Agonist stimulation of the FZD 6 -G protein complex is followed by rapid dissociation (Kilander et al, 2014b). The FZD isoform, FZD 4 , on which this study focuses has so far not been connected to signaling through heterotrimeric G proteins.…”

“…In contrast to transmembrane receptors, intracellular proteins, such as the heterotrimeric G proteins, are not directly affected by surface CL in this assay. The mobile fraction of intracellular proteins is reduced only upon interaction with CL-immobilized surface proteins (Qin et al, 2008(Qin et al, , 2011Kilander et al, 2014b). In cells transfected with fluorescently tagged FZD 4 , Ga, and untagged bg, we found that the mobile fraction of FZD 4 was remarkably reduced by surface cross-linking (Fig.…”

“…On the other hand, random collision coupling of G protein and receptors is sufficient to form the fully active, agonist-bound receptor conformation inducing release of GDP from Ga and subsequently a rapid GTP-dependent dissociation of the receptor-G protein complex (Neubig, 1994;Hein et al, 2005;Oldham and Hamm, 2008;Rasmussen et al, 2011;Ayoub et al, 2012); both proposed scenarios are supported experimentally (Galés et al, 2005(Galés et al, , 2006Hein et al, 2005;Nobles et al, 2005;Qin et al, 2011). FZD 6 exists in an inactive-state complex with heterotrimeric Ga i and Ga q (Kilander et al, 2014b) similar to what was observed for the muscarinic M 3 receptor (Qin et al, 2011). Agonist stimulation of the FZD 6 -G protein complex is followed by rapid dissociation (Kilander et al, 2014b).…”

“…The procedure was essentially as described in Kilander et al (2014b), Qin et al (2011), andQin et al (2008). Cells were grown on 35-mm extracellular matrix; coated (1:300; Sigma-Aldrich) glass-bottom dishes and assessed using a Zeiss 710 laser-scanning microscope (Zeiss, Jena, Germany).…”

“…1). To assess FZD 4 -G protein interaction, we took advantage of a dual-color FRAP (dcFRAP) protocol that enables simultaneous assessment of lateral mobility of two fluorescently tagged proteins (Phair et al, 2004;Qin et al, 2008Qin et al, , 2011Dorsch et al, 2009;Kilander et al, 2014b) (Fig. 1).…”