Abstract

Frizzleds (FZDs) are unconventional G protein–coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD–G protein coupling remain poorly understood. Previously, we showed that FZD₆ assembles with Gα_i1/Gα_q (but not with Gα_s, Gα_o and Ga_12/13), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD₄, a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD₄ assembles—in a DVL-independent manner—with Gα_12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Gα_i1, Gα_o, Gα_s, and Gα_q. The FZD₄–G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD₄ green fluorescent protein–transfected cells depend on Gα_12/13. Furthermore, expression of FZD₄ and Gα₁₂ or Gα₁₃ in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Gα_12/13 signaling, underlining the functionality of an FZD₄-Gα_12/13-RHO signaling axis. In summary, Gα_12/13-mediated WNT/FZD₄ signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD₄ signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.