Post-translational Proteolytic Processing of the Calcium-independent Receptor of α-Latrotoxin (CIRL), a Natural Chimera of the Cell Adhesion Protein and the G Protein-coupled Receptor

Abstract

The calcium-independent receptor of ␣-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys-and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and twosubunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.Cell adhesion receptors provide physical links between cell plasma membranes and the extracellular matrix. These receptors have large extracellular domains that contain characteristic structural modules that are directly involved in cell-to-cell and cell-to-matrix interaction. They can also function as signaling receptors, providing the cell with critical information required for proper tissue growth and development. The signaling function of cell adhesion receptors has been primarily attributed to their connection to the tyrosine phosphorylation pathway via tyrosine kinase or phosphatase domains in their intracellular region or to the recruitment of other tyrosine kinases upon activation. The recent discovery of heptahelical receptors with large extracellular cell adhesion-like domains opens an intriguing possibility that cell-to-cell and cell-to-matrix interaction can be also coupled to G protein signaling (1, 2).By this mechanism, cells should be able to produce fast and sensitive responses to physical contacts with the extracellular environment.Putative cell adhesion GPCRs 1 are linked to different cellular functions in a variety of tissues. CD97 and EMR1 (F4/80 antigen) are involved in leukocyte activation (3-6). The calcium-independent receptor of ␣-latrotoxin (CIRL), a neuronal target of a presynaptic neurotoxin, has been implicated in the regulation of secretion (7,8). BAI, a p53-inducible protein, is an inhibitor of angiogenesis (9). The Drosophila receptor Flamingo has a role in establishing planar cell polarity, as pointed out by genetic studies (10). Expression of the HE6 receptor is restricted to epididymis, thus suggesting its function in sperm maturation (11). Most of the other adhesion GPCRs were discovered by gene sequencing and have not been thoroughly characterized either functionally or biochemically. Among the adhesion GPCRs, only CD97 has a known binding partner, the membrane protein CD55 (or d...

Vasculostatin, a proteolytic fragment of Brain Angiogenesis Inhibitor 1, is an antiangiogenic and antitumorigenic factor

“…The GPS domain is found by homology in 20 otherwise functionally unrelated G-protein-coupled receptors, and proteolytic processing of this domain has been shown for a subset of these receptors (Stacey et al, 2000). The protease that recognizes the GPS domain and cleaves at this specific site is unknown (Krasnoperov et al, 2002). So far, BAI1 is the only protein for which proteolytic processing at the GPS domain releases an extracellular fragment with antiangiogenic properties.…”

“…To examine the role of the GPS domain in the cleavage of BAI1, we constructed a specific point mutation converting the conserved serine at the P1 0 site to an alanine. Point mutation of this conserved serine/threonine to an alanine has been previously shown to result in abrogation of cleavage at the GPS site for other receptors (Krasnoperov et al, 2002). Transfection of wild-type (w.t) and the S 927 -A 927 BAI1 into 293 cells yielded full-length (170 kDa) protein in the cell lysate (Figure 1f).…”

“…We searched the predicted protein sequence of BAI1 using Simple Modular Architecture Research Tool (SMART) for consensus sequence of known proteolytic cleavage sites and identified a G-protein-coupled receptor proteolytic cleavage site (GPS) at the junction of the N-terminal extracellular region with the first transmembrane domain (Figure 1a). GPS domains are approximately 50 residues long containing either two or four conserved cysteinecs and are found by sequence homology in otherwise functionally unrelated receptors including latrophilin-1 (CL1), hCD97, Flamingo-1, sea urchin sperm receptor for egg jelly (suREJ3) and Ig Hepta (Gray et al, 1996;Krasnoperov et al, 2002;Mengerink et al, 2002). Alignment of the GPS domains in BAI1 and other GPS-containing receptors is shown in Figure 1c.…”

“…Alignment of the GPS domains in BAI1 and other GPS-containing receptors is shown in Figure 1c. The arrowhead above the sequences indicates the putative cleavage site within this domain (Krasnoperov et al, 2002).…”

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor

“…A long N-terminal extracellular domain characterizes the LNB GPCRs, featuring functional motifs present in other proteins, where they appear to be important for protein-protein or adhesion-type interactions (15,17). The vast majority of members of this family remain orphans, and whether these receptors signal through a conventional G protein mechanism is largely unknown (18).…”