Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins

Abstract

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channnels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related with adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to a impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts

“…In addition, based on the identification of the interactome of GlialCAM and MLC1, we recently proposed that MLC1 could act as a tetraspanin-like molecule [ 16 ]. Tetraspanins are cell-surface proteins with four transmembrane domains, which can homo- and hetero-oligomerize.…”

“…Unsurprisingly, several transporters and ion channels were identified by different groups (for review [ 13 ]). In an extensive proteomic analysis [ 16 ], we have recently identified the following interactor proteins: the chloride channel ClC-2, the gap junction protein Cx43, the glutamate transporter EAAT1/2, the alpha2 and beta2 subunits of the sodium/potassium ATPase, the sodium bicarbonate transporter NBCe1, the glucose transporter GLUT1, and the sodium calcium exchanger. Furthermore, although not appearing as MLC interacting proteins, other activities have been shown to be affected by the lack of MLC1 such as the volume regulated anion channel (VRAC) or the calcium-permeable channel TRPV4.…”

“…As described previously, defects in either the MLC1 or GlialCAM interacting proteins’ function might also affect extracellular potassium dynamics. Astrocyte uptake is mainly mediated by the Na + /K + ATPase pump [ 45 ], an interactor of MLC1 [ 16 , 18 , 19 ]. In Mlc1 KO mice, there is a reduced expression of the inward rectifier potassium channel Kir4.1 involved in potassium clearance [ 35 ], whose absence is known to lead to hyperexcitability and epilepsy [ 46 ].…”

“…Even though the function of the MLC1/GlialCAM complex is unknown, it has been hypothesized that it may have a role in the regulation of ion/water homeostasis, as it interacts with different transporters and ion channels. Thus, it has been shown that the complex interacts directly with the chloride channel 2 (ClC-2) [ 14 ], the gap junction alpha 1 protein (connexin 43, Cx43) [ 15 , 16 , 17 ], Na + /K + -ATPase [ 18 , 19 ], and it is thought to regulate indirectly the activity of volume-regulated anion channel (VRAC) [ 10 , 20 , 21 ] or the calcium-permeable channel TRPV4 [ 22 ]. Recent bioinformatics developments have suggested new potential functions for the GlialCAM/MLC1 complex (see Section 2 ).…”

Knockdown of GPRC5B alleviates the high glucose-induced inflammation and extracellular matrix deposition of podocyte through inhibiting NF-κB pathway

“…8 It is significantly up-regulated in common human glomerulopathy (including DN, immunoglobulin A (IgA) nephropathy, and lupus nephritis) and can activate NF-κB pathway. 7 In addition, overexpression of GPRC5B in the fibrotic heart, lung, and liver can enhance the production of collagen in myofibroblasts, thus directly promoting fibrosis in tissues. 9 However, the specific role of GPRC5B in DN is unclear.…”