Abstract
ABSTRACTAdgra2, formerly known as Gpr124, is a key regulator of cerebrovascular development in vertebrates. Together with the GPI-anchored glycoprotein Reck, this adhesion GPCR (aGPCR) stimulates Wnt7-dependent Wnt/β-catenin signaling to promote brain vascular invasion in an endothelial cell-autonomous manner. Adgra2 and Reck have been proposed to assemble a receptor complex at the plasma membrane, but the molecular modalities of their functional synergy remain to be investigated. In particular, as typically found in aGPCRs, the ectodomain of Adgra2 is rich in protein-protein interaction motifs whose contributions to receptor function are unknown. In opposition to the severe ADGRA2 genetic lesions found in previously generated zebrafish and mouse models, the zebrafish ouchless allele encodes an aberrantly-spliced and inactive receptor lacking a single leucine-rich repeat (LRR) unit within its N-terminus. By characterizing this allele we uncover that, in contrast to all other extracellular domains, the precise composition of the LRR domain determines proper receptor trafficking to the plasma membrane. Using CRISPR/Cas9 engineered cells, we further show that Adgra2 trafficking occurs in a Reck-independent manner and that, similarly, Reck reaches the plasma membrane irrespective of Adgra2 expression or localization, suggesting that the partners meet at the plasma membrane after independent intracellular trafficking events.
Highlights
Adhesion G protein-coupled receptors constitute the second largest group of GPCRs in humans
Adgra2ouchless accumulates within the endoplasmic reticulum The adgra2 variant found in ouchless mutants differs from adgra2 reference sequences by four non-synonymous SNPs as well as a 72 bp deletion corresponding to exon 4 (Fig. 1A)
Adgra2 and Reck are recently recognized synergistic activators of Wnt7-stimulated Wnt/β-catenin signaling acting in CNS-invading endothelial cell (EC) and neural crest-derived cells of the zebrafish dorsal root ganglia (DRG). They have been proposed to contribute to the assembly of a Wnt7 receptor complex at the plasma membrane operating either as a stand-alone receptor complex or in association with the classical Fzd/Lrp5/6 receptors (Noda et al, 2016; Vanhollebeke et al, 2015), but very limited information is far available on their mechanism(s) of action
Summary
Adhesion G protein-coupled receptors (aGPCRs) constitute the second largest group of GPCRs in humans. Most aGPCRs are orphan receptors with no identified ligands that function through remarkably diverse mechanisms (Fredriksson et al, 2003; Hamann et al, 2015) They differ from other GPCRs by long N-terminal extensions preceding a membrane-proximal GPCR autoproteolysisinducing (GAIN) domain containing the highly conserved GPCR proteolytic site (GPS) (Araç et al, 2012). Vascularization and blood-brain barrier maturation are impaired in all or parts of the zebrafish and mouse central nervous system, respectively (Anderson et al, 2011; Cullen et al, 2011; Kuhnert et al, 2010; Vanhollebeke et al, 2015) This receptor promotes angiogenic sprouting through endothelial cell (EC)-autonomous Wnt/β-catenin signaling stimulation upon contact with neural progenitor-derived Wnt ligands (Posokhova et al, 2015; Vanhollebeke et al, 2015; Zhou and Nathans, 2014)
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