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The β1-adrenergic receptor (β1AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β1AR-M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R3.50 - D/E6.30 "ionic lock"). Here we compare eight new structures of β1AR-M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of β-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R3.50 - E6.30 distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in β1AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation. [ABSTRACT FROM AUTHOR]

In previous work we described six point mutations that thermostabilised the turkey β1-adrenergic receptor (tβ1AR). The thermostable mutant, tβ1AR-m23, had an apparent Tm 21°C higher than the native protein when solubilized in dodecylmaltoside (DDM) and, in addition, was significantly more stable in short chain detergents, which allowed its crystallization and structure determination. Identification of thermostabilizing mutations in tβ1AR was performed by systematic mutagenesis followed by expressing and assaying each of the 318 mutants for their thermostability. This is time-consuming, so to facilitate studies on related receptors, we have studied the transferability of these mutations to the human adrenergic receptors, hβ1AR and hβ2AR, which have, respectively, 76% and 59% sequence identity to tβ2AR, excluding the N- and C-termini. Thermostability assays revealed that hβ1AR was much more unstable than tβ2AR, whereas hβ2AR was more stable than tβ1AR. Addition of the 6 thermostabilizing mutations in tβ2AR-m23 into both hβ2AR and hβ2AR increased their apparent Tms by 17°C and 11°C, respectively. In addition, the mutations affected the global conformation of the human receptors so that they were predominantly in the antagonist bound form, as was originally observed for tβ2AR-m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. [ABSTRACT FROM AUTHOR]

There are ≈350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermo-stable mutants of a GPCR, the β1-adrenergic receptor. The most stable mutant receptor, βAR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, βAR-m23 was as stable as bovine rhodopsin. In addition. βAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand. [ABSTRACT FROM AUTHOR]

It has been shown that sera from chagasic patients with positive EVI serology could act in co-operation with complement or normal human lymphocytes as a partial β-adrenoceptor agonist increasing the contractile tension and frequency of isolated rat atria, as occurs with IgG purified from chagasic serum. In this paper we demonstrate that IgG present in chagasic patients sera could bind to the β-adrenoceptors of the heart and stimulate contractile activity of myocardium. The positive inotropic and chronotropic effect could be blocked by the specific β1-adrenoceptor antagonist but not by the β2-adrenoceptor antagonist. Chagasic IgG inhibited the binding of (-) ⊃3H-DHA to β-adrenoceptors of purified rat myocardial membranes behaving as non-competitive inhibitors. The reactivity of chagasic serum or IgG with β1-adrenoceptor was lost after absorptions with turkey red blood cells. In contrast, guinea-pig red blood cells were unable lo remove the β1 reactivity of chagasic serum or chagasic IgG. This supports the specificity of β1-adrenoceptors of the chagasic IgG and the independence of β1-adrenoceptor reactivity in relation to the EVI system. Clinical specificity of the β1-adrenoceptor reactivity seems rather high in Chagas' disease since it was lacking in 14 individuals with other cardiopathies, such as ischaemic and rheumatic heart disease, even after heart surgery. [ABSTRACT FROM AUTHOR]

Abstract: Structural studies on G-protein-coupled receptors have been hampered for many years by their instability in detergent solution and by the number of potential conformations that receptors can adopt. Recently, the structures of the β1 and β2 adrenergic receptors and the adenosine A2a receptor were determined in the antagonist-bound state, a receptor conformation that is thought to be more stable than the agonist-bound state. In contrast to these receptors, the neurotensin (NT) receptor NTS1 is much less stable in detergent solution. We have therefore used a systematic mutational approach coupled with activity assays to identify receptor mutants suitable for crystallization, both alone and in complex with the peptide agonist NT. The best receptor mutant NTS1-7m contained four point mutations. It showed increased stability compared to the wild-type receptor, in the absence of ligand, after solubilization with a variety of detergents. In addition, NTS1-7m bound to NT was more stable than unliganded NTS1-7m. Of the four thermostabilizing mutations, only one residue (A86L) is predicted to be in the lipid environment. In contrast, I260A appears to be buried within the transmembrane helix bundle, F342A may form a distant part of the putative ligand-binding site, whereas F358A is likely to be in a region that is important for receptor activation. NTS1-7m binds NT with a similar affinity for the wild-type receptor. However, agonist dissociation was slower, and NTS1-7m activated G-proteins poorly. The affinity of NTS1-7m for the antagonist SR48692 was also lower than that of the wild-type receptor. Thus, we have successfully stabilized NTS1 in an agonist-binding conformation that does not efficiently couple to G-proteins. [Copyright &y& Elsevier]