Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of ␣ subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-␣ ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-␣ among a range of substrates tested. Furthermore, HIF-␣ subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-␣ interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-␣ molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-␣, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the ␤-domain of pVHL and loss of interaction was associated with defective HIF-␣ ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-␣ proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its ␤ domain interacts with short recognition sequences in HIF-␣ subunits.

Using multivalent protein probes, an evolutionarily conserved endogenous ligand for EMR2, a human myeloid cell-restricted EGF-TM7 receptor, was identified on the surface of a number of adherent cell lines. In addition, in situ staining of the ligand has revealed specific in vivo patterns consistent with a connective tissue distribution. The interaction is conserved across species and mediated exclusively by the largest EMR2 isoform containing 5 epidermal growth factor (EGF)-like modules. Antibody-blocking studies subsequently revealed that the fourth EGF-like module constitutes the major ligandbinding site. The largest isoform of CD97, a related EGF-TM7 molecule containing an identical EGF-like module, also binds to the putative EMR2 ligand. Through the use of mutant Chinese hamster ovary (CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as the enzymatic removal of specific cell surface GAGs, the molecular identity of the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS GAGs blocked the EMR2-ligand interaction in a dose-dependent manner. EMR2-CS interaction is Ca 2؉ -and sulphation-dependent and results in cell attachment. This is the first report of a GAG ligand for the TM7 receptors extending the already vast repertoire of stimuli of the GPCR superfamily.

Loss-of-function mutations in the gene encoding G proteincoupled receptor 56 (GPR56) lead to bilateral frontoparietal polymicrogyria (BFPP), an autosomal recessive disorder affecting brain development. The GPR56 receptor is a member of the adhesion-GPCR family characterized by the chimeric composition of a long ectodomain (ECD), a GPCR proteolysis site (GPS), and a sevenpass transmembrane (7TM) moiety. Interestingly, all identified BFPP-associated missense mutations are located within the extracellular region of GPR56 including the ECD, GPS, and the extracellular loops of 7TM. In the present study, a detailed molecular and functional analysis of the wild-type GPR56 and BFPP-associated point mutants shows that individual GPR56 mutants most likely cause BFPP via different combination of multiple mechanisms. These include reduced surface receptor expression, loss of GPS proteolysis, reduced receptor shedding, inability to interact with a novel protein ligand, and differential distribution of the 7TM moiety in lipid rafts. These results provide novel insights into the cellular functions of GPR56 receptor and reveal molecular mechanisms whereby GPR56 mutations induce BFPP.

EMR2 is a human myeloid-restricted member of the EGF-TM7 receptor family that contains a highly conserved G protein-coupled receptor proteolysis site (GPS) in the membrane-proximal region. Here the post-translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leu 517 -Ser 518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular K K-subunit and transmembrane L L-subunit requires a minimum of eight amino acids in the L L-subunit. The GPS motif is necessary, but not su⁄cient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures.

GPR56/ADGRG1 is a versatile adhesion G protein-coupled receptor with diverse biological functions. GPR56 expression is variably detected in human melanoma cell lines and correlates inversely with the metastatic potential of melanoma lesions. GPR56 associates with the tetraspanins CD9 and CD81 on the melanoma cell surface. GPR56 activation by immobilized CG4 monoclonal antibody facilitates N-terminal fragment dissociation in a CD9/CD81-dependent manner specifically inducing IL-6 production, which promotes cell migration and invasion. Interestingly, expression of GPR56-C-terminal fragment alone recapitulates the antibody-induced receptor function, implicating a major role for the C-terminal fragment in GPR56 activation and signaling. Analysis of site-directed mutant receptors attests the importance of the conserved N-terminal residues of the C-terminal fragment for its self-activation. Finally, we show that the GPR56-induced signaling in melanoma cells is mediated by the Gα/RhoA pathway. In summary, the expression and activation of GPR56 may modulate melanoma progression in part by inducing IL-6 production after N-terminal fragment dissociation and C-terminal fragment self-activation.

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EMR2/ADGRE2 is a human myeloid-restricted adhesion G protein-coupled receptor critically implicated in vibratory urticaria, a rare type of allergy caused by vibration-induced mast cell activation. In addition, EMR2 is also highly expressed by monocyte/macrophages and has been linked to neutrophil migration and activation. Despite these findings, little is known of EMR2-mediated signaling and its role in myeloid biology. In this report, we show that activation of EMR2 via a receptor-specific monoclonal antibody promotes the differentiation of human THP-1 monocytic cell line and induces the expression of pro-inflammatory mediators, including IL-8, TNF-α, and MMP-9. Using specific signaling inhibitors and siRNA knockdowns, biochemical and functional analyses reveal that the EMR2-mediated signaling is initiated by Gα16, followed by the subsequent activation of Akt, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and nuclear factor kappa-light-chain-enhancer of activated B cells. Our results demonstrate a functional role for EMR2 in the differentiation and inflammatory activation of human monocytic cells and provide potential targets for myeloid cell-mediated inflammatory disorders.