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.

Specific expression of GPR56 by human cytotoxic lymphocytes.

We here report the existence of a new cluster of adhesion-GPCRs in human immune cells. Analysis of a comprehensive immune cell transcriptome dataset indicated that expression of the closely related receptors, GPR56, GPR97, and GPR114, is associated with single lymphocyte and granulocyte subsets. Applying flow cytometric analysis with newly generated mAb, we show that expression of GPR56 is restricted to cytotoxic NK and T lymphocytes, including CD8(+), CD4(+), and γδ T cells. Primary infection with human CMV, which generates a vast population of CD8(+) T cells with an effector phenotype, induced a strong increase in GPR56 expression in virus-specific CD8(+) T cells that remained detectable during latency. In NK-92 cells, ectopic expression of GPR56 inhibited spontaneous and SDF-1-stimulated cell migration. Our data suggest that GPR56 expression is a common trait of human cytotoxic lymphocytes and might affect the migratory properties of these cells.

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.