GPR124 regulates murine CNS angiogenesis and blood-brain barrier formation by an intracellular domain-independent mechanism

The GPR124/RECK/WNT7 pathway essentially regulates central nervous system angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion 7-pass transmembrane protein, associates with membrane RECK, which binds and stabilizes newly synthesized WNT7 for subsequent transfer to Frizzled (FZD) and canonical b-catenin signaling. GPR124 function remains enigmatic; while its extracellular domain (ECD) is required, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially mediated by bridging of the GPR124 and FZD ICDs by intracellular adaptor proteins. GPR124 ICD deletion mutants impair zebrafish angiogenesis, but paradoxically still mediate WNT7 signaling upon mammalian cell transfection. We thus further investigated the GPR124 C-terminal ICD by deletion in mice (Gpr124ΔC). Notably, Gpr124ΔC/ΔC mice could be born and did not recapitulate Gpr124-/- embryonic lethal forebrain hemorrhage. GPR124ΔC protein was inefficiently expressed, resulting in mild, hypomorphic, non-hemorrhagic defects in CNS angiogenesis and BBB function, sparing the cortex and only confined to ganglionic eminences. Impaired surface expression of GPR124ΔC directly correlated with reduced endothelial WNT signaling, arguing against an intrinsic signaling deficiency. Further, GPR124ΔC and recombinant GPR124 ECD both rescued WNT7 signaling following brain endothelial Gpr124 knockdown. Thus, in mice, the GPR124 essential regulation of WNT7-dependent CNS forebrain angiogenesis and BBB function is exerted by ICD-independent functionality, extending the range of signaling mechanisms used by adhesion 7-pass transmembrane receptors.