Article

Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif

Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2004; 279(30):31823-32. DOI: 10.1074/jbc.M402974200
Source: PubMed

ABSTRACT Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu downward arrow Ser(518) cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P(+1) cleavage site (Ser(518)) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P(-2) His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.

Download full-text

Full-text

Available from: James Harris, Aug 21, 2015
0 Followers
 · 
117 Views
  • Source
    • "Therefore, it's very likely that Vlgr1 could undergo self-cleavage that separates the protein into two non-covalent associated subunits, if the M 5890 sits in a similar hydrophobic pocket as the Leu 837 in CL-1 receptor (Arac et al. 2012). The proteolytic cleavage of transmembrane proteins have multiple functions, such as activation of the precursor protein of ENAC, inactivation of V2 vasopressin receptor, and particularly, separation of the LNB7TM GPCRs such as GPR56 and EMR2 precursor into two non-covalent subunits (Kleyman et al. 2009; Kojro and Fahrenholz 1995; Lin et al. 2004; Paavola et al. 2011). GPS site cleavage is required for EMR2 mediated inflammatory cytokine production in macrophages and the cleaved two subunits localize both as hetero-dimer and monomer (Huang et al. 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The very large G protein coupled receptor (Vlgr1) is a member of adhesion receptors or large N-terminal family B-7 transmembrane helixes (LNB7TM) receptors within the seven trans-membrane receptor superfamily. Vlgr1 is the largest GPCR identified to date; its mRNA spans 19 kb and encodes 6,300 amino acids. Vlgr1 is a core component of ankle-link complex in inner ear hair cells. Knock-out and mutation mouse models show that loss of Vlgr1 function leads to abnormal stereociliary development and hearing loss, indicating crucial roles of Vlgr1 in hearing transduction or auditory system development. Over the past 10 or so years, human genetics data suggested that Vlgr1 mutations cause Usher syndromes and seizures. Although significant progresses have been made, the details of Vlgr1's function in hair cells, its signaling cascade, and the mechanisms underlying causative effects of Vlgr1 mutations in human diseases remain elusive and ask for further investigation.
    Journal of Molecular Neuroscience 11/2012; 50(1). DOI:10.1007/s12031-012-9911-5 · 2.76 Impact Factor
  • Source
    • "GPS cleavage would generate two separate protein bands (cleaved and noncleaved) that would be detected by both antibodies, whereas cleavage-deficiency results in a single noncleavaged protein fraction. As controls, we used a similar EMR2 construct (Lin et al., 2004), a mammalian aGPCR known to undergo GPS proteolysis, and a GPS-mutated EMR2 variant which is resistant to autoproteolytic cleavage (Chang et al., 2003). Both control transgenes were only C-terminally mFc-tagged but did not contain an N-terminal HA-tag. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. Results: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115, and Gpr116. We show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111, or Gpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. Conclusions: The aGPCR cluster Gpr110, Gpr111, Gpr115, and Gpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115. Developmental Dynamics 241:1591-1602, 2012. © 2012 Wiley Periodicals, Inc.
    Developmental Dynamics 10/2012; 241(10):1591-602. DOI:10.1002/dvdy.23841 · 2.67 Impact Factor
  • Source
    • "However, other studies, including that of Araç et al, have shown that this is not always the case (Qian et al, 2002). More likely, mutations in the core of the GAIN domain lead to protein folding defects, which would in turn impair trafficking (Lin et al, 2004). Autoproteolysis may therefore represent a mechanism by which the GAIN domain is locked into its functional and presumably more stable state after proper folding occurs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A large and poorly understood class of G protein-coupled receptors (GPCRs) are involved in cell adhesion and contain an autoproteolytic site known as the GPCR proteolysis site (GPS) located immediately N-terminal to the first transmembrane span. This motif of similar to 50 amino acids is also found juxtaposed to the first transmembrane span of an unrelated family of proteins associated with polycystic kidney disease (PKD), but its structural and functional roles were not clear. In this issue of The EMBO Journal, Arac et al use X-ray crystallography to show that the GPS motif is merely the C-terminal end of a much larger (G) under bar PCR (a) under bar utoproteolysis (in) under bar ducing (GAIN) domain. Atomic models for two of these ancient domains allow one to map the sites of mutations associated with cancer or PKD, and hint at functional roles other than autoproteolysis.
    The EMBO Journal 03/2012; 31(6):1334-5. DOI:10.1038/emboj.2012.51 · 10.75 Impact Factor
Show more