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

Abstract

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.

G‐protein activation by putative antagonists at mutant Thr373Lys α2A adrenergic receptors

“…Partial agonist activity of antagonists has been reported (Cho et al, 1996;Groblewski et al, 1997) induction of partial agonist properties of the AT 2 -speci®c peptide ligand CGP 42112A [27% of the response to the agonist (Sar 1 )AII] is associated with an increased a nity for the mutant AT 1A angiotensin II receptor (Groblewski et al, 1997). Thus, with the exception of CGP 42112A, partial agonist activity was not accompanied by an increased binding a nity at this latter receptor.…”

The Myocardium-protective Gly-49 Variant of the β1-Adrenergic Receptor Exhibits Constitutive Activity and Increased Desensitization and Down-regulation

“…The marked amplification of the response to the agonist and the moderate changes of K d and K act values are less common, but they were reported for some GPCRs with constitutive activity, such as the Asn-111 mutant of the AT 1A receptor (17).…”

The angiotensin II type 1 receptor and receptor-associated proteins

“…B a s e d o n a n u m b e r o f pharmacological, mutagenesis and computer-oriented modeling studies [63][64][65][66][67][68][69][70][71][72][73][74][75][76], the general agreement is that a positively-charged residue of Lys199 on transmembrane domain (TM)-5 of the AT1 receptor interacts with the -carboxyl group of Phe8 in Ang II. This interaction is crucial for high affinity binding of Ang II to the receptor.…”