Molecular insights into Adgra2/Gpr124 and Reck intracellular trafficking

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.

Protocol publication

[…] Images were acquired on a Zeiss LSM710 confocal microscope. Colocalization was assessed using the Pearson correlation coefficient (PCC) calculated with coloc2 in ImageJ (NIH). Applied to colocalization analysis, PCC is a measure of the linear correlation between the intensities observed in the two channels of interest. A value of 1 indicates that the two signals are positively and perfectly linearly related; a value of −1 indicates a perfectly linear, but negative correlation; and a value close to zero indicates that the distributions of the two signals are uncorrelated. [...] Statistical analyses were performed using the GraphPad Prism software. Sample size was determined with the G*power 3.1.5 software to reach adequate statistical power. Each dot plot value represents an independent cell or embryo and every experiment was conducted three times independently. P-values were calculated by the Kruskal–Wallis test (post hoc Dunn's test). […]