The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important for this interaction (Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, A. V., Ullrich, A., and Bö hmer,

Rat bradykinin B 2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B 2 receptor was isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA) 30 -mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B 2 receptor showed phosphorylation at Bradykinin, a member of the kinin family (1), is a nonapeptide with diverse biological activities ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3). Bradykinin mediates its physiological effects by binding to and activation of the bradykinin B 2 receptor. Molecular cloning has revealed the primary structure of the B 2 receptor (4) and classified it as a member of the G protein-coupled receptor superfamily. The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is still rather little direct evidence at the protein level for attributes such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B 2 or other G protein-coupled receptors. Indeed phosphorylation sites for few G protein-coupled receptors have been mapped to date (10 -12), and most of these sites reflect the in vitro phosphorylation of the isolated receptor.We report here on the isolation of rat B 2 receptor from transfected Chinese hamster ovary (CHO) cells using oligo(dA) covalently linked to bradykinin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out under very mild conditions using oligo(dT) columns analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically separated receptor, subsequent peptide mass fingerprinting by matrix-assisted laser desorption...

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Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.