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

Yi-Shu Huang,Nien-Yi Chiang,Gin-Wen Chang,Hsi-Hsien Lin

doi:10.1038/s41598-018-22849-x

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.

Highlights

Characterized by a long extracellular domain (ECD) with cell-adhesion functions and a seven-transmembrane (7TM) domain with signaling functions, the adhesion G protein-coupled receptors have been implicated in diverse biological activities and human diseases[1]
In order to discriminate the membrane-associated and hetero-dimeric N-terminal fragment (NTF) of aGPCRs36,37, we analyzed two previously-described chimeric EMR2 receptors (EMR2-WT-PAR1-myc and EMR2-S518A-PAR1-myc) containing the EMR2-NTF fused with the protease-activated receptor 1 (PAR1) in which the GPCR proteolysis site (GPS) site is located upstream of the thrombin site (Fig. 1A)
An apparent size difference was noted between the thrombin-released hetero-dimeric NTF and the one remained on the membrane

Summary

Introduction

Characterized by a long extracellular domain (ECD) with cell-adhesion functions and a seven-transmembrane (7TM) domain with signaling functions, the adhesion G protein-coupled receptors (aGPCRs) have been implicated in diverse biological activities and human diseases[1]. Not all aGPCRs undergo GPS proteolysis and non-cleavable aGPCRs are still able to be activated[30] Both Stachel-dependent and -independent activation mechanisms have been shown and GPS proteolysis was shown in a recent publication functionally unnecessary for the cleavable Drosophila latrophilin (dCIRL) to initiate cellular mechano-signaling[17,31,32,33]. Regardless, the current consensus is that the strength and mode of interaction between the NTF and CTF seem to play a key role in modulating the activation and signaling activities of aGPCRs30,34 In this context, of interest is an alternative aGPCR activation mechanism, the split personality hypothesis, whereby the NTF and CTF are dissociated but remain as independent entities on the membrane[35]. Our findings shed lights on a divergent NTF form of aGPCRs and its functional implication in modulating aGPCR activities is discussed

Methods

Results

Conclusion