Heparin interacts with the adhesion GPCR GPR56, reduces receptor shedding, and promotes cell adhesion and motility.

GPR56 is an adhesion-class G-protein-coupled receptor responsible for bilateral frontoparietal polymicrogyria (BFPP), a severe disorder of cortical formation. Additionally, GPR56 is involved in biological processes as diverse as hematopoietic stem cell generation and maintenance, myoblast fusion, muscle hypertrophy, immunoregulation and tumorigenesis. Collagen III and tissue transglutaminase 2 (TG2) have been revealed as the matricellular ligands of GPR56 involved in BFPP and melanoma development, respectively. In this study, we identify heparin as a glycosaminoglycan interacting partner of GPR56. Analyses of truncated and mutant GPR56 proteins reveal two basic-residue-rich clusters, R(26)GHREDFRFC(35) and L(190)KHPQKASRRP(200), as the major heparin-interacting motifs that overlap partially with the collagen III- and TG2-binding sites. Interestingly, the GPR56-heparin interaction is modulated by collagen III but not TG2, even though both ligands are also heparin-binding proteins. Finally, we show that the interaction with heparin reduces GPR56 receptor shedding, and enhances cell adhesion and motility. These results provide novel insights into the interaction of GPR56 with its multiple endogenous ligands and have functional implications in diseases such as BFPP and cancer.

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding.

Disease-associated GPR56 mutations cause bilateral frontoparietal polymicrogyria via multiple mechanisms.

Loss-of-function mutations in the gene encoding G protein-coupled 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 seven-pass 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.

GPS proteolytic cleavage of adhesion-GPCRs.

The stability and functional diversity of proteins can be greatly modulated by posttranslational modification. Proteolytic cleavage at the GPCR proteolysis site (GPS) has been identified as an intrinsic protein modification process of many adhesion-GPCRs. In recentyears, the conserved cleavage site, molecularmechanism and the potential functional implication of the GPS proteolysis have been gradually unveiled. However, many aspects of this unique cleavage reaction including its regulation, the relationship between the cleaved fragments and the functional pathways mediated by the cleaved receptor subunits, remain unanswered. Further investigation of the GPS proteolytic modification shall shed light on the biology of the adhesion-GPCRs.

Overexpression of FAM46A, a Non-canonical Poly(A) Polymerase, Promotes Hemin-Induced Hemoglobinization in K562 Cells.

FAM46A belongs to the FAM46 subfamily of the nucleotidyltransferase-fold superfamily and is predicted to be a non-canonical poly(A) polymerase. FAM46A has been linked to several human disorders including retinitis pigmentosa, bone abnormality, cancer, and obesity. However, its molecular and functional characteristics are largely unknown. We herein report that FAM46A is expressed in cells of the hematopoietic system and plays a role in hemin-induced hemoglobinization. FAM46A is a nucleocytoplasmic shuttle protein modified by Tyr-phosphorylation only in the cytosol, where it is closely associated with ER. On the other hand, it is located proximal to the chromatin regions of active transcription in the nucleus. FAM46A is a cell cycle-dependent poly-ubiquitinated short-lived protein degraded mostly by proteasome and its overexpression inhibits cell growth and promotes hemin-induced hemoglobinization in K562 cell. Site-directed mutagenesis experiments confirm the non-canonical poly(A) polymerase activity of FAM46A is essential for enhanced hemin-induced hemoglobinization. In summary, FAM46A is a novel poly(A) polymerase that functions as a critical intracellular modulator of hemoglobinization.

Multivalent protein probes for the identification and characterization of cognate cellular ligands for myeloid cell surface receptors.

Cell-cell interactions mediated by cell surface receptor-ligand pairs in the immune system are often of low affinity and transient in nature. To begin to study these weak interactions, it is desirable to devise a generally applicable method for screening for and enriching cells expressing low-affinity ligands for specific cell surface receptors. We describe here an experimental strategy that uses a multivalent form of protein as a probe to identify and characterize cognate ligand(s) of myeloid cell surface receptors. Recombinant fusion proteins containing the receptor protein fragment of interest fused to a truncated Fc domain and a unique biotinylation signal are produced, biotinylated, and coupled to (strep)avidin-coated fluorescent or paramagnetic microspheres. These multivalent microparticle probes are then used to screen or capture cells expressing the cognate cellular ligand(s).

Putative alternative trans-splicing of leukocyte adhesion-GPCR pre-mRNAs generates functional chimeric receptors.

The EGF-TM7 receptors, a subfamily of adhesion-GPCRs mostly restricted to leukocytes, are known to express multiple functional protein isoforms through extensive alternative cis-splicing. Here, we demonstrate that EGF-TM7 pre-mRNAs also undergo the rare trans-splicing, leading to the generation of functional chimeric receptors. RT-PCR and in silico analyses of EMR2 transcripts identified unique fragments containing the EGF-like motif 3 of a closely related EGF-TM7 gene, CD97, in addition to the alternative cis-spliced products. The sequence swapping is restricted to the EGF-3 exon, generating unique EMR2(1-2-3*-5) and EMR2(1-2-3*-4-5) molecules, which are functional in ligand-binding as the wild-type EMR2(1-2-3-4-5) and CD97(1-2-3-4-5) receptors. Our results suggest that human leukocytes employ trans-splicing as well as cis-splicing to increase the repertoire of functional adhesion-GPCRs.

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

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.

High levels of soluble GPR56/ADGRG1 are associated with positive rheumatoid factor and elevated tumor necrosis factor in patients with rheumatoid arthritis.

BACKGROUND: GPR56/ADGRG1 is a member of the adhesion-class G protein-coupled receptor (aGPCR) family important in brain development, oncogenesis and tumor metastasis. Like other aGPCRs, GPR56 is cleaved at the GPCR proteolysis site (GPS) motif into an N-terminal fragment (NTF) and a C-terminal fragment (CTF). Existence of soluble GPR56 (sGPR56) has been shown in vitro, however the underlying mechanism and its pathophysiologic role remains undetermined. OBJECTIVE: To assess the presence of sGPR56 in human serum using ELISA assay and compare the serum sGPR56 levels among patients of various chronic inflammatory diseases and healthy subjects. PATIENTS AND METHODS: In this study, serum samples from patients with systemic lupus erythematosus (SLE) (n = 57), rheumatoid arthritis (RA) (n = 95), Sjögren's syndrome (SS) (n = 29), ankylosing spondylitis (AS) (n = 51), and normal controls (n = 81) were analyzed using sGPR56-specific ELISA. RESULT: We show that serum sGPR56 levels are increased in patients of RA, but not in those with SLE, SS and AS. Intriguingly, serum sGPR56 levels in RA patients correlated with positive rheumatoid factor, a marker of bone erosion and poor outcome. In addition, an elevated sGPR56 level is also noted in RA patients with higher tumor necrosis factor level. CONCLUSION: we conclude that sGPR56 is present in vivo and sGPR56 level is elevated in certain chronic inflammatory diseases such as RA. Hence, sGPR56 might be considered a potential biomarker for RA disease progression.

GPR56/ADGRG1 Activation Promotes Melanoma Cell Migration via NTF Dissociation and CTF-Mediated Gα12/13/RhoA Signaling.

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 Gα12/13/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.

Activation of Adhesion GPCR EMR2/ADGRE2 Induces Macrophage Differentiation and Inflammatory Responses via Gα16/Akt/MAPK/NF-κB Signaling Pathways.

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.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells.

The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular alpha part and a TM7/cytoplasmic beta part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16(+) blood monocytes, on macrophages, and on BDCA-3(+) myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.

Proteolytic cleavage of the EMR2 receptor requires both the extracellular stalk and the GPS motif.

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 Leu517-Ser518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular alpha-subunit and transmembrane beta-subunit requires a minimum of eight amino acids in the beta-subunit. The GPS motif is necessary, but not sufficient 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.

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.

The 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 self-catalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (α) and a seven-pass transmembrane subunit (ß) with cellular adhesion and signaling functions, respectively. Using the myeloid cell-restricted 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 α-ß 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.

GPS autoproteolysis is required for CD97 to up-regulate the expression of N-cadherin that promotes homotypic cell-cell aggregation.

Most adhesion-class G protein-coupled receptors (adhesion-GPCRs) undergo a novel self-catalytic cleavage at the GPCR proteolysis site (GPS) to form a hetero-dimeric complex containing the extracellular and seven-span transmembrane subunits. However, little is known about the role of GPS auto-proteolysis in the function of adhesion-GPCRs. Here we show that GPS cleavage is essential for the homotypic cell aggregation promoted by CD97 receptor, a leukocyte-restricted adhesion-GPCR often aberrantly expressed in carcinomas. We find that CD97 does not mediate cell aggregation directly. Instead, expression of the wild type - but not the GPS cleavage-deficient CD97 up-regulates the expression of N-cadherin, leading to Ca(++)-dependent cell-cell aggregation. Our results provide a clear evidence for the role of GPS proteolytic modification in the cellular function of adhesion-GPCRs.

Leukocyte adhesion-GPCR EMR2 is aberrantly expressed in human breast carcinomas and is associated with patient survival.

EGF-like module containing mucin-like hormone receptor 2 (EMR2) is a leukocyte-restricted adhesion G protein-coupled receptor. Aberrant expression of EMR2 and its highly homologous molecule CD97 have been reported in various human cancers. Herein, we investigate the expression of EMR2 in neoplastic breast human tissue and its relationship with patient survival. EMR2 expression in normal and neoplastic breast tissue was assessed by immunohistochemistry in sections from 10 normal controls and micro-arrayed tissue cores from 69 cases of ductal carcinoma in situ (DCIS) and 272 invasive carcinomas. The pattern and intensity of staining was correlated with the clinicopathological characteristics of each case and the disease outcome. While absent in normal breast epithelium, EMR2 was significantly up-regulated in the cytoplasmic and nuclear compartments of both DCIS and invasive carcinoma, with invasive samples displaying significantly higher expression levels compared with in situ disease. In invasive disease, EMR2 cytoplasmic expression was significantly associated with higher tumour grade but not with patient age, nodal status, tumour size, estrogen receptor expression, relapse-free or overall survival. In contrast, EMR2 nuclear expression correlated negatively with higher tumour grade. Of note, EMR2 nuclear expression was associated with longer relapse-free survival as well as overall survival. This study indicates that EMR2 is expressed in neoplastic breast epithelium and suggests that expression patterns of EMR2 are relevant in breast cancer progression. The association of improved patient survival with higher nuclear expression levels identifies EMR2 as a potential biomarker in patients with invasive breast cancer.

Site-specific N-glycosylation regulates the GPS auto-proteolysis of CD97.

Auto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark of adhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders, information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97, a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incompletely processed, unlike its close homolog, epidermal growth factor-like module-containing mucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of related adhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-specific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggest that N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production of either cleaved or uncleaved molecules.

The role of receptor oligomerization in modulating the expression and function of leukocyte adhesion-G protein-coupled receptors.

The human leukocyte adhesion-G protein-coupled receptors (GPCRs), the epidermal growth factor (EGF)-TM7 proteins, are shown here to function as homo- and hetero-oligomers. Using cell surface cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer analysis of EMR2, an EGF-TM7 receptor predominantly expressed in myeloid cells, we demonstrate that it forms dimers in a reaction mediated exclusively by the TM7 moiety. We have also identified a naturally occurring but structurally unstable EMR2 splice variant that acts as a dominant negative modulator by dimerizing with the wild type receptor and down-regulating its expression. Additionally, heterodimerization between closely related EGF-TM7 members is shown to result in the modulation of expression and ligand binding properties of the receptors. These findings suggest that receptor homo- and hetero-oligomerization play a regulatory role in modulating the expression and function of leukocyte adhesion-GPCRs.