Membrane-association of EMR2/ADGRE2-NTF is regulated by site-specific N-glycosylation

Abstract

The evolutionarily conserved adhesion G protein-coupled receptors (aGPCRs) play critical roles in biological processes as diverse as brain development, cell polarity and innate immune functions. A defining feature of aGPCRs is the GPCR autoproteolysis inducing (GAIN) domain capable of self-catalytic cleavage, resulting in the generation of an extracellular N-terminal fragment (NTF) and a seven-transmembrane C-terminal fragment (CTF) involved in the cellular adhesion and signaling functions, respectively. Interestingly, two different NTF subtypes have previously been identified, namely an NTF that couples non-covalently with the CTF and a membrane-associated NTF that tethers on cell surface independently. The two NTF subtypes are expected to regulate aGPCR signaling via distinct mechanisms however their molecular characteristics are largely unknown. Herein, the membrane-associated NTF of EMR2/ADGRE2 is investigated and found to be modified by differential N-glycosylation. The membrane association of EMR2-NTF occurs in post-ER compartments and site-specific N-glycosylation in the GAIN domain is involved in modulating its membrane-association ability. Finally, a unique amphipathic α-helix in the GAIN domain is identified as a putative membrane anchor of EMR2-NTF. These results provide novel insights into the complex interaction and activation mechanisms of aGPCRs.

Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs)

“…We suggest that for both proteins, there must be a mechanism that is independent of interaction with their TM domains that allows an excess of the ectodomain to remain bound to the exosome after the ultracentrifugation steps. One idea is that there may be an amphipathic α-helix in the ectodomain of PC1 which allows the N-terminus to remain associated with the ELV membrane similar to that seen in EMR2/ADGRE2, a protein with a GPS/GAIN domain 32 . We were unable to detect such an in-plane membrane anchor using AmphipaSeeK in PC1 33 .…”

G Protein-Coupled Receptors in the Sweet Spot: Glycosylation and other Post-translational Modifications

“…The GPS cleavage takes place in the ER and can be affected by N-glycosylation as reported for CD97 antigen . Similarly, the membrane association of the N-terminal fragment of EMR2, resulting from cleavage, was shown to be dependent on site-specific N-glycosylation …”

“…73 Similarly, the membrane association of the N-terminal fragment of EMR2, resulting from cleavage, was shown to be dependent on site-specific N-glycosylation. 74 Among rhodopsin-type GPCRs, PARs are the most thoroughly characterized receptors, the function and signaling of which is regulated by proteolytic cleavage. All four receptors (PAR1−4) have either predicted or detected O-glycosylation sites in close proximity to the cleavage sites.…”

Adhesion G-protein coupled receptors: Implications for metabolic function

“…The GAIN domain is a unique evolutionarily conserved structure of aGPCRs that is required for self-cleavage (Prömel et al, 2013). The N and C termini re-associate upon maturation and remain non-covalently attached to each other at the The capacity of the N-terminal fragment of an aGPCR to cross-assemble with the Cterminus of a different aGPCR and vice versa, and the ability of a small proportion of the Nterminal segment, anchored independently to the cell membrane, to initiate downstream signalling upon ligand binding, increases the complexity of aGPCR heterodimer signalling considerably (Volynski et al, 2004;Silva et al, 2009;Huang et al, 2018). The GPS is found immediately upstream of the first transmembrane loop of aGPCRs and it is rich in cysteine residues, alternating with tryptophan in a C-W-W-C-C conserved sequence (Araç et al, 2012).…”