Proximity Interactome Analysis of Super Conserved Receptors Expressed in the Brain Identifies EPB41L2, SLC3A2, and LRBA as Main Partners.

The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the ß-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.

Sulf2a controls Shh-dependent neural fate specification in the developing spinal cord.

Sulf2a belongs to the Sulf family of extracellular sulfatases which selectively remove 6-O-sulfate groups from heparan sulfates, a critical regulation level for their role in modulating the activity of signalling molecules. Data presented here define Sulf2a as a novel player in the control of Sonic Hedgehog (Shh)-mediated cell type specification during spinal cord development. We show that Sulf2a depletion in zebrafish results in overproduction of V3 interneurons at the expense of motor neurons and also impedes generation of oligodendrocyte precursor cells (OPCs), three cell types that depend on Shh for their generation. We provide evidence that Sulf2a, expressed in a spatially restricted progenitor domain, acts by maintaining the correct patterning and specification of ventral progenitors. More specifically, Sulf2a prevents Olig2 progenitors to activate high-threshold Shh response and, thereby, to adopt a V3 interneuron fate, thus ensuring proper production of motor neurons and OPCs. We propose a model in which Sulf2a reduces Shh signalling levels in responding cells by decreasing their sensitivity to the morphogen factor. More generally, our work, revealing that, in contrast to its paralog Sulf1, Sulf2a regulates neural fate specification in Shh target cells, provides direct evidence of non-redundant functions of Sulfs in the developing spinal cord.