Activation of Myeloid Cell-Specific Adhesion Class G Protein-Coupled Receptor EMR2 via Ligation-Induced Translocation and Interaction of Receptor Subunits in Lipid Raft Microdomains

Abstract

fThe adhesion class G protein-coupled receptors (adhesion-GPCRs) play important roles in diverse biological processes ranging from immunoregulation to tissue polarity, angiogenesis, and brain development. These receptors are uniquely modified by selfcatalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (␣) and a seven-pass transmembrane subunit (␤) with cellular adhesion and signaling functions, respectively. Using the myeloid cellrestricted EMR2 receptor as a paradigm, we exam the mechanistic relevance of the subunit interaction and demonstrate a critical role for GPS autoproteolysis in mediating receptor signaling and cell activation. Interestingly, two distinct receptor complexes are identified as a result of GPS proteolysis: one consisting of a noncovalent ␣-␤ heterodimer and the other comprising two completely independent receptor subunits which distribute differentially in membrane raft microdomains. Finally, we show that receptor ligation induces subunit translocation and colocalization within lipid rafts, leading to receptor signaling and inflammatory cytokine production by macrophages. Our present data resolve earlier conflicting results and provide a new mechanism of receptor signaling, as well as providing a paradigm for signal transduction within the adhesion-GPCR family.T he adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR subfamily, whose 33 members are expressed restrictedly in cells of the central nervous, immune, and/or reproductive systems (2, 53). Adhesion-GPCRs are uniquely characterized by the chimeric composition of a large extracellular domain (ECD) and a seven-pass transmembrane (7TM) region. While the 7TM region is predicted to transduce cellular signals, the ECD of adhesion-GPCRs contains multiple repeats of protein modules such as the lectin-like, immunoglobulin (Ig)-like, epidermal growth factor (EGF)-like, and cadherinlike motifs known to mediate protein-protein interaction (2, 53). Adhesion-GPCRs are thus thought to possess a dual cellular adhesion and signaling function. Recent studies have revealed many important functions for adhesion-GPCRs: these include development of the brain frontal cortex (34), circulation of cerebrospinal fluid (44), central nervous system (CNS)-restricted angiogenesis and vascularization (1, 10, 21), myelination of Schwann cells (30, 31), Usher syndrome (29, 49), cellular polarity (16, 23), epididymal fluid regulation and male fertility (4, 12), and immune recognition and regulation (11,18,27,47), as well as tumor growth and metastasis (8,17,43,50). However, the molecular mechanisms mediating the biological functions of adhesion-GPCRs remain to be fully characterized.In addition to the large mosaic ECD, the complex pre-and posttranslational modifications that produce multiple receptor isoforms and the lack of defined ligands also present a great challenge in deciphering the molecular mechanisms of adhesion-GPCRs. Of note is the conserved prot...

Shear Stress–Dependent Downregulation of the Adhesion-G Protein–Coupled Receptor CD97 on Circulating Leukocytes upon Contact with Its Ligand CD55

“…As a consequence of the two-subunit structure of aGPCRs, the two chains can behave as independent proteins as has been shown by studies with transfected cell lines for latrophilin and EMR2 (55,56). In this study, we failed to demonstrate an independent existence of the transmembrane b-chain of CD97 after dissociation from the extracellular a-chain.…”

CD97 inhibits cell migration in human fibrosarcoma cells by modulating TIMP‐2/MT1‐ MMP/MMP‐2 activity – role of GPS autoproteolysis and functional cooperation between the N‐ and C‐terminal fragments

“…For example, receptor cleavage was shown to be critical for EMR2-mediated functions, but not essential for latrophilin signaling activity in Caenorhabditis elegans [33,34]. Moreover, the two cleaved receptor subunits were reported to either work together as a noncovalent heterodimeric complex or independently as separate entities [33]. In some cases, the NTF can be shed or cleaved, resulting in a CTF that, alone, delivers higher signaling activity than the full-length wild-type receptor [35,36].…”

“…GPS proteolysis is required for the maturation and surface expression of some but not all adhesion-GPCRs. For example, receptor cleavage was shown to be critical for EMR2-mediated functions, but not essential for latrophilin signaling activity in Caenorhabditis elegans [33,34]. Moreover, the two cleaved receptor subunits were reported to either work together as a noncovalent heterodimeric complex or independently as separate entities [33].…”

“…Horseradish peroxidase-conjugated secondary antibody and mouse IgG 1 isotype control were from Sigma-Aldrich and R&D Systems, respectively. Antibodies that recognize EMR2 (2A1 for the NTF, and a rabbit polyclonal antiserum for the CTF) and CD97 (CLB/CD97-1) have been described previously [33,37]. All culture media were from Invitrogen (Carlsbad, CA, USA), and were supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 IUÁmL À1 penicillin, and 50 lgÁmL À1 streptomycin.…”

“…Generation of HT1080 cells stably overexpressing recombinant CD97 receptors HT1080 cells stably expressing CD97-WT and cleavage-deficient S531A isoforms as well as the EMR2(EGF125) isoform were generated as described previously [21,33]. For the generation of stable HT1080 cells overexpressing various recombinant CD97 receptors, the relevant CD97 constructs were engineered in the pFB-Neo vector by the use of appropriate primers (Table 1) with standard molecular biology techniques.…”