Frizzled4 (FZD4) is a receptor for Norrin and WNT7A/B ligands, is expressed in endothelial cells (ECs), is required for blood-CNS-barrier function as well as CNS angiogenesis, and transduces β-catenin-dependent signaling. Despite its fundamental importance in neurovascular biology, including as drug target, the molecular mechanisms governing FZD4 regulation remain poorly understood. Here, we employed proximity biotinylation to identify proteins that regulate FZD4. We identified ATPase Plasma Membrane Ca2+ Transporting 1 (ATP2B1, also known as PMCA1) as a FZD4 proximity interactor. Functional analyses revealed that ATP2B1 depletion increased EC Ca2+ and significantly attenuated Norrin/Frizzled4 signaling. These effects of Atp2b1 deficiency were recapitulated by ionomycin-mediated elevation of intracellular Ca2+ and suppressed by inhibition of calcineurin/NFAT signaling. Endothelial-specific Atp2b1 deletion caused retinal vascular defects consistent with compromised Norrin/Frizzled4 signaling. In the developing brain, WNT7A/B pathway LOF phenotypes in Gpr124 KO mice were exacerbated by additional deletion of Atp2b1 in ECs. This study identifies ATP2B1 as a novel regulator of Norrin- and WNT7A/B-induced FZD4 signaling and suggests that in pathological contexts with elevated EC Ca2+-levels, EC function may be modulated by suppression of β-catenin-dependent signaling. In addition, this mechanism may be relevant in the context of pharmacological approaches that transiently open the barrier for drug delivery.
