Sequence analysis predicts three and purified from the growth medium by metal affinity chromatography followed by size exclusion chromatography (SEC)

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Sequence analysis predicts three and purified from the growth medium by metal affinity chromatography followed by size exclusion chromatography (SEC). binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane. Introduction An important feature of nervous system development is the process in which axons elongate and navigate through a complex cellular embryonic terrain to locate their appropriate synaptic partners, which may be millimeters or even many centimeters away (1). The sensing organ of the growing axon is the growth cone, a specialized motile tip of the axon that is responsible for sensing the local environment and PD168393 moving toward the neuron’s target cell (2). Axons often elongate alongside other axons, forming bundles or fascicles, in a process known as fasciculation. Upon reaching their target region, growth cones steer away from each other to innervate their own specific targets, in a process called defasciculation (3). Cell adhesion molecules (CAMs), which are found on the surface of axons, have been implicated in mediating axon fasciculation and defasciculation. Two such CAMsneurotactin (Nrt) and amalgam (Ama)have been identified in (4) that is responsible for specifying the correct head and thoracic segmental identity of the embryo. Schneider 2 (S2) cells transfected with the gene secrete the protein into the medium. Use of this medium in a cell aggregation assay showed that Ama possesses heterophilic adhesion properties and serves as a ligand DSTN for Nrt (5). Aggregation assays performed with Ama that had been engineered to be expressed on the plasma membrane of S2 cells showed that it also possesses homophilic adhesion properties (6). Nrt is a type-II transmembrane glycoprotein that functions as a heterophilic cell adhesion molecule in axon path-finding and fasciculation (7C9). It has a 324-amino acid NH2-terminal cytoplasmic domain that has been assigned to the class of intrinsically disordered proteins (10), a transmembrane sequence, and a 500-amino-acid COOH-terminal cholinesterase-like ectodomain. Over the course of embryogenesis, Nrt is expressed in proliferating and differentiating cells, and is first expressed in early embryogenesis at the onset of cellularization. During cellularization, it is localized on the growing plasma membrane (11,12). This recruitment to specific regions of the plasma membrane suggests that Nrt may PD168393 bind to cytoskeleton components. Based on the adhesion properties of both Nrt and Ama, it has been suggested that Ama serves as a linker protein between Nrt-expressing cells, and that Nrt-mediated adhesion may be considered as a two-step process: 1) binding of Ama to the ectodomain; and 2) stabilization and strengthening of the interaction via clustering of Nrt at sites of cell-cell contact, an interaction that requires the presence of the cytoplasmic domain of Nrt and binding to cytoskeletal components (5). It has been shown that mutations in Ama dominantly enhance the mutant phenotype of Abelson tyrosine kinase (Abl) (6). The cytoplasmic tyrosine kinase Abl is incorporated into multiple signaling networks, including those that regulate cell cycle progression, cytoskeletal dynamics, and axon outgrowth (13). In gene encodes a 333-amino-acid polypeptide, and its sequence indicates that Ama is a secreted member of the immunoglobulin PD168393 superfamily (IgSF). It has an NH2-terminal signal sequence, three immunoglobulin (Ig) domains, and a short COOH-terminal segment. IgSF members often contain more than one Ig domain; indeed, Ama consists of three tandem Ig domains that are 18C32% identical to each other in pairwise comparisons. Ama shares 26% overall sequence identity and 42% sequence similarity with rat NCAM 1; in particular, the positions of the two cysteines that form a conserved disulfide bond and of a Trp that is part of the hydrophobic core are conserved in all three Ama domains. Sequence analysis predicts three and purified from the growth medium by metal affinity chromatography followed by size exclusion chromatography (SEC). Ama was deglycosylated by Endo F1 as previously described (17). Analytical ultracentrifugation data acquisition and?analysis Analytical ultracentrifugation sedimentation equilibrium (AUC-SE) analysis was performed using a Beckman Optima XLA instrument (Beckman Coulter, Fullerton, CA) in which the protein concentration distribution within the cell was monitored by its absorbance at 260 nm. The protein concentration was 0.45 mg/mL in twofold concentrated phosphate-buffered saline. Data were acquired over 3 days using six-sector cells in an AnTi 50 rotor with column heights of 12 mm, at rotor speeds of 14,000, 17,000 and 20,000 rpm, until equilibrium had been reached at each speed, as shown by the perfect overlay of runs measured at 2 h intervals. Data.