Jean-Claude Bonnafous

Mutation of Asn111 in the Third Transmembrane Domain of the AT1A Angiotensin II Receptor Induces Its Constitutive Activation

A preliminary model of the rat AT1A angiotensin II (AII) receptor (Joseph, M. P., Maigret, B., Bonnafous J.-C., Marie, J., and Scheraga, H. A. (1995) J. Protein Chem. 14, 381-398) has predicted an interaction between Asn111 located in transmembrane domain (TM) III and Tyr292 (TM VII) in the nonactivated receptor; a disruption of this interaction upon AII activation would allow Tyr292 to interact with the conserved Asp74 (TM II). The previous verification that Tyr292 is essential for receptor coupling to phospholipase C (Marie, J., Maigret, B., Joseph, M. P., Larguier, R., Nouet, S., Lombard, C., and Bonnafous, J.-C. (1994) J. Biol. Chem. 269, 20815-20818) prompted us to check the possible alterations in receptor properties upon Asn111 → Ala mutation. The mutated receptor (N111A) displayed: (i) strong constitutive activity, with amplification of the maximal phospholipase C response to AII; (ii) agonist behavior of the AT2-specific ligand CGP 42112A, [Sar1,Ile8]AII, and [Sar1,Ala8]AII, antagonists of the wild-type receptor; (iii) inverse agonism behavior of the non-peptide ligands DuP 753, LF 7-0156, and LF 8-0129. The results are discussed in the light of the allosteric ternary complex models and other described examples of constitutive activation of G protein-coupled receptors.

A computer modeling postulated mechanism for angiotensin II receptor activation

The angiotensin II receptor of the AT1-type has been modeled starting from the experimentally determined three-dimensional structure of bacteriorhodopsin as the template. Intermediate 3D structures of rhodopsin andβ2-adrenergic receptors were built because no direct sequence alignment is possible between the AT1 receptor and bacteriorhodopsin. Docking calculations were carried out on the complex of the modeled receptor with AII, and the results were used to analyze the binding possibilities of DuP753-type antagonistic non-peptide ligands. We confirm that the positively charged Lys199 on helix 5 is crucial for ligand binding, as in our model; the charged side chain of this amino acid interacts strongly with the C-terminal carboxyl group of peptide agonists or with the acidic group at the 2′-position of the biphenyl moiety of DuP753-type antagonists. Several other receptor residues which are implicated in the binding of ligands and the activation of receptor by agonists are identified, and their functional role is discussed. Therefore, a plausible mechanism of receptor activation is proposed. The three-dimensional docking model integrates most of the available experimental observations and helps to plan pertinent site-directed mutagenesis experiments which in turn may validate or modify the present model and the proposed mechanism of receptor activation.

The kinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin: an antagonist of the angiotensin AT1 receptor which also binds to the AT2 receptor.

1 Agonists and antagonists of kinin B1 and B2 receptors were evaluated in vitro for their effects against angiotensin II (AII)‐induced contractile responses in the rabbit aorta and for their binding properties to angiotensin AT1 and AT2 receptors from purified membrane of rat liver and lamb uterus respectively. 2 In aortic rings, the kinin B1 receptor antagonist, des‐Arg9‐[Leu8]bradykinin (BK) (3–100 μm) caused a concentration‐dependent decrease in sensitivity and a depression of the maximum response to AII. Des‐Arg10‐[Leu9]kallidin (KD), des‐Arg9‐BK, des‐Arg10‐KD, BK or KD at 3 μm had no effect against All‐induced contractions. 3 Des‐Arg9‐[Leu8]BK (3 or 100 μm) did not affect contractions of aortic rings to histamine, potassium chloride, endothelin‐1, 5‐hydroxytryptamine, noradrenaline and the thromboxane A2‐mimetic, U46619. 4 Des‐Arg9‐[Leu8]BK displaced [125I]‐Sar1‐AII binding to the AT1 subtype in rat liver membranes with a Ki value of 1.1 ± 0.4 μm. Values of Ki for des‐Arg9‐BK and KD were 45 ± 13 μm and 25 ± 22 μm, respectively. The other kinin derivatives des‐Arg10‐KD, BK and des‐Arg10‐[Leu9]KD at concentrations up to 100 μm did not bind to the AT1 receptor. 5 All the kinin derivatives except BK bound to AT2 receptors in lamb uterus membranes. Values of Ki for des‐Arg9‐[Leu8]BK, des‐Arg10‐[Leu9]KD, des‐Arg9‐BK, des‐Arg10‐KD and KD were 0.3 ± 0.1, 0.7 ± 0.1, 1.2 ± 0.3, 1.5 ± 0.3 and 7.0 ± 1.6 μm, respectively. 6 In conclusion, des‐Arg9‐[Leu8]BK is an insurmountable antagonist of All‐induced contractions in the rabbit aorta and also binds with a relatively high affinity to AT1 and AT2 receptors in isolated membrane fractions. These additional properties of des‐Arg9‐[Leu8]BK should be considered when it is used as an antagonist to characterize kinin B1 receptors.

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.

Design of Angiotensin II Derivatives Suitable for Indirect Affinity Techniques: Potential Applications to Receptor Studies

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

Characterization of the Angiotensin II Receptor

We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.