High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line.

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.

Techniques: Oxidative cross-linking as an emergent tool for the analysis of receptor-mediated signalling events.

The development of methods for investigating protein-protein interactions in native complexes and in living cells is an important goal in proteomics. Chemical cross-linking represents a potentially powerful approach to this goal. In this article, we review the application of recently developed oxidative cross-linking techniques to this problem; the involved reactions, mediated by high-valent metal-chelate complexes, are highly efficient in many cases, extremely rapid and do not require chemically modified proteins, although new chemical design strategies can broaden the scope of applications. Some promising applications of this chemistry to model systems is reviewed, including the fast and convenient covalent labelling of G-protein-coupled receptors (GPCRs) in intact cells, with an emphasis on the perspectives of mapping signalling events triggered by these complexes. Progress towards resolving the outstanding problems of signalling network elucidation, in addition to chemical and analytical issues that must be addressed, are discussed.

Crosslinking photosensitized by a ruthenium chelate as a tool for labeling and topographical studies of G-protein-coupled receptors.

The purpose was to apply oxidative crosslinking reactions to the study of recognition and signaling mechanisms associated to G-protein-coupled receptors. Using a ruthenium chelate, Ru(bipy)(3)(2+), as photosensitizer and visible light irradiation, in the presence of ammonium persulfate, we performed fast and efficient covalent labeling of the B(2) bradykinin receptor by agonist or antagonist ligands possessing a radio-iodinated phenol moiety. The chemical and topographical specificities of these crosslinking experiments were investigated. The strategy could also be applied to the covalent labeling of the B(1) bradykinin receptor, the AT(1) angiotensin II receptor, the V(1a) vasopressin receptor and the oxytocin receptor. Interestingly, we demonstrated the possibility to covalently label the AT(1) and B(2) receptors with functionalized ligands. The potential applications of metal-chelate chemistry to receptor structural and signaling studies through intramolecular or intermolecular crosslinking are presented.

Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol covalent attachment sites on estrogen receptor alpha.

Mass spectrometry was used to identify the sites of covalent attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The covalent attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxyl or counterpart hydroxyl with histidine 524 of the human estrogen receptor alpha.

We investigated the role of H524 of the human estrogen receptor alpha (ERalpha) for the binding of various estrogens [estradiol (E(2)), 3-deoxyestradiol (3-dE(2)), and 17beta-deoxyestradiol (17beta-dE(2))] and antiestrogens [4-hydroxytamoxifen (OHT), RU 39 411 (RU), and raloxifene (Ral)], which possess the 17beta-hydroxyl or counterpart hydroxyl (designated: 17beta/c-OH), with the exception of 17beta-dE(2) and OHT. The work involved a comparison of the binding affinities of these ligands for wild-type and H524 mutant ERs, modified or not with diethyl pyrocarbonate (DEPC), a selective histidine reagent. Alanine substitution of H524 did not significantly change the association affinity constant (relative to OHT) of 17beta-dE(2), whereas those of RU, Ral, E(2), and 3-dE(2) were decreased 3-fold, 14-fold, 24-fold, and 49-fold, respectively. Values of the two ligands available in radiolabeled form (E(2) and OHT) were correlated with the dissociation rate constants, which were increased 250-fold and 2-fold, respectively. The action of DEPC on wild-type ER led to a homogeneous ER population which still bound antiestrogens and 17beta-dE(2) with practically unchanged affinities (less than 4-fold decreases in relative affinity constants), while E(2) and 3-dE(2) displayed markedly decreased affinities (56-fold decrease for E(2)). Conversely, DEPC treatment of H524A mutant ER did not induce marked decreases in the relative affinities of any of the checked compounds (decreases wild-type ER) and very weakly protected H524A ER. Molecular modeling was tentatively used to interpret the biochemical results.