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.

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Available from: James Harris, Aug 21, 2015
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    • "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). "
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    Journal of Molecular Neuroscience 11/2012; 50(1). DOI:10.1007/s12031-012-9911-5 · 2.76 Impact Factor
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    • "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. "
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    Developmental Dynamics 10/2012; 241(10):1591-602. DOI:10.1002/dvdy.23841 · 2.67 Impact Factor
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    • "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. "
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