Constitutive activation of G protein-coupled receptors, initially reported by Cotecchia et al. (1) for the ␣ 1 -adrenergic receptor, is now well documented and has been extended to many other members of this large family (2-21). Moreover, mutations inducing receptor constitutive activation have been found to be associated with human diseases (13-16). Data on the ␤ 2 -adrenergic receptors have prompted their authors to propose an extended version of the ternary complex model, based on the existence of active and inactive receptor states (4,22). The same mechanistic model, either in its initial form (22) or in a refined version (23), have provided interpretations for the "negative antagonism" or "inverse agonism" phenomenon, evidenced for ligands of ␤ 2 -adrenergic (24, 25), B 2 bradykinin (26), m5 muscarinic (21), and thyrotropin-releasing hormone (18) receptors. As emphasized by the recent work of Cotecchia's group on the ␣ 1B -adrenergic receptor (8), a correlation between mechanistic considerations and molecular events associated to receptor conformational changes is obviously required. The work reported in the present paper is based on a previous molecular modeling study (27) which aimed at predicting modifications of specific amino acid side chain interactions during the process of the angiotensin II type 1 (AT 1 ) 1 receptor activation. This preliminary model (27) interaction in the transduction mechanism (28). The model prompted us to check whether the absence of Asn 111 would favor this interaction, leading to constitutive receptor activity.In this paper we demonstrate that the N111A mutant receptor displays a strong constitutive activity as well as striking pharmacological changes. The results are discussed in the light of above-mentioned current models (22, 23). MATERIALS AND METHODS Reagents Site-directed Mutagenesis and ExpressionThe amino acid mutation Asn 111 3 Ala was carried out as described previously (28). The cDNA sequences of the wild type and N111A mutant rat AT 1A receptors were subcloned in the XbaI site of the polylinker of the eukaryotic expression vector pCMV (31). Receptors were transiently expressed in COS-7 cells by using the electroporation transfection method: 10 7 cells were resuspended in 300 l of electroporation buffer (50 mM K 2 HPO 4 , 20 mM CH 3 COOK, 20 mM KOH, pH 7.40) and incubated for 10 min at room temperature in an electroporation cuvette (0.4-cm electrode gap, Bio-Rad) with 20 g of pCMV carrier and different amounts of pCMV containing cDNA receptors sequences (30 -300 ng range). They were submitted to an electric discharge (950 microfarads, 280 V, 50 ms), then cultured for 2 days at 37°C in Dulbecco's modified Eagle's medium, 4.5 g/liter glucose, 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin before binding or IP accumulation experiments.

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 and beta 2-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.

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We report that mutation of specific residues in the human B2 bradykinin (BK) receptor induces its marked constitutive activation, evaluated through inositol phosphate production in COS-7 cells expressing the wild-type or mutant receptors. We provide evidence for a strikingly high constitutive activation of the B2 receptor induced by alanine substitution of the Asn113 residue, located in the third transmembrane domain. These results are reminiscent of our previous finding that mutation of the homologous Asn111 residue induces constitutive activation of the AT1 angiotensin II receptor. BK overstimulation of the constitutively activated mutant N113A receptor was also observed. Phe replacement of the Trp256 residue, fairly conserved in transmembrane domain VI of G protein-coupled receptors, also induced a less prominent but significant constitutive activation. Interestingly, the peptidic HOE 140 compound and an original nonpeptidic compound LF 16 0335, which both behaved as inverse agonists of the wild-type receptor expressed in COS-7 cells, became potent and efficient agonists of the two constitutively activated mutant N113A and W256F receptors. These parallel changes observed for two chemically unrelated series can serve as a basis for future studies of structure-function relationships and modeling of activation processes, based on a detailed analysis of the network of helix-helix interactions, which stabilize the inactive receptor conformation and undergo rearrangements on transition to activated states.