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...
Although it is clear that positive selection of T cells involves recognition of specific self-peptide/MHC complexes, the nature of these self-ligands and their relationship to the cognate antigen are controversial. Here we used two complementary strategies to identify naturally occurring self-peptides able to induce positive selection of T cells bearing a specific T cell receptor, OT-I. Both the bioassay- and bioinformatics-based strategies identified the same self-peptides, derived from F-actin capping protein and beta-catenin. These peptides displayed charge conservation at two key TCR contact residues. The biological activity of 43 other self-peptides and of complex peptide libraries directly correlated to the extent of conservation at TCR contact residues. These results demonstrate that selecting self-peptides are rare and can be identified by homology-based search strategies.
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.
GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.Covalent modification by acetylation or fatty acylation occurs on a variety of viral and cellular proteins (1). N-terminal acetylation is one of the most common protein modifications, occurring on ϳ85% of eukaryotic proteins (2). The amino acid residue adjacent to the amino-terminal methionine residue determines whether the N-terminal methionine is retained or removed before acetylation (2). Proteins that contain the Nterminal sequence MGXXX(S/T) undergo a different set of modifications. The initiating Met is removed, and myristate is added to the N-terminal glycine. The requirement for glycine at the N terminus is absolute for N-myristoylation to occur.In contrast to N-terminal acetylation and myristoylation, which occur co-translationally, palmitoylation is a post-translational lipid modification. Nearly all palmitoylated proteins are S-acylated by attachment of palmitate via a thioester linkage to the sulfhydryl group of cysteine. Exceptions include adenylate cyclase toxin from Bordetella pertussis, which is modified by amide-linked palmitoylation on the ⑀-amino group of lysine residues (3) and human sonic hedgehog, which is palmitoylated through an amide linkage to the N-terminal cysteine (4). Unlike myristoylation, the enzymology of palmitoylation reactions is poorly understood. Palmitoylacyl transferases have not been thoroughly purified and characterized. Moreover, no study has directly examined the nature of thioester-linked palmitoylation in vivo at a molecular level. All of the studies on thioester-linked palmitoylation are based on incorporation of radioactive palmitate. The stoichiometry of palmitoylation as well as the molecular identity of the attached fatty acid moiety (palmitate a...
A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein.
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