Identification of residues required for ligand binding to the beta-adrenergic receptor.

Strader, Catherine D.; Sigal, Irving S.; Register, R B; Candelore, Mari R.; Rands, Elaine; Dixon, Richard A. F.

doi:10.1073/pnas.84.13.4384

Cited by 372 publications

(222 citation statements)

References 19 publications

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“…Its mutation with serine in the P-adrenergic receptor yielded two smaller imrnunoreactive polypeptides [20]. A tenfold reduction in rhodopsin levels has been observed in the nina A mutant of Drosophila melanogaster [71].…”

Section: Discussionmentioning

confidence: 99%

“…inter-or intra-residue distances, the resulting dNN distances between amide protons of adjacent residues in the (2-6) and (10)(11)(12)(13)(14) regions are close to the ideal 0.27-nm value, whereas d,, distances between CaH,-NH,,, range over 0.31 -0. 35 20.01 nm (ideal value for a-helical structure 0.36 nm). The systematic 30% increase (0.07 nm to 0.10 nm) of all dnN and d, , distances found when protons from the side chains of Asn8 or Tyrl2 were taken as internal references may thus be related to difference in the mobility of side-chains and backbone protons [61].…”

Section: Residuementioning

confidence: 99%

See 1 more Smart Citation

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Berlose

Convert

Brunissen

et al. 1994

European Journal of Biochemistry

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The S/T-XI-X,-N-P-X,-X,-Y highly conserved sequence of the seventh transmembrane (TM VII) segment of G-protein-coupled receptors is not present in the photon receptor bacteriorhodopsin TM VII domain. Despite this noticeable discrepancy in sequence, the X-ray structure of bacteriorhodopsin is generally used as the key structure for modelling all G-protein-coupled receptors. Thus, a kinked truns-Pro helix is usually accepted for the TM VII three-dimensional structure of G-proteincoupled receptors, although Asn-Pro dipeptide mainly induces a type UIII /3-turn conformation in both model peptides and proteins. NMR studies in various solvents and molecular calculations were undertaken in order to gain insight into the conformational behaviour of a 15-residue peptide from the tachykinin NK-1 TM VII domain incorporating this common sequence. The low solubility of this membrane-embedded peptide precludes methanol or micellar systems mimicking membrane environment; thus only dimethylsulfoxide (Me,SO) or chlorofonn/Me,SO mixture could be used. We also found that perfluoro-tert-butanol, which has not been previously used for NMR studies, constitutes an excellent alternative solvent for the analysis of hydrophobic peptides. The postulated kinked truns-Pro helix was only present as a minor conformer in Me,SO and an equilibrium between helical and extended structures existed. From NOE data a type I/III ,&structure, centered around Pro9-Ile10, probably stabilized by an Asx turn, may be postulated. Addition of chloroform in Me,SO increased the percentage of folded structures but no preferential conformation could be proposed. In perfluoro-tert-butanoI/CD,OD (9 : 1) the N-and C-terminal regions presented an a-helical structure, and these two domains were linked by a hinge around Asn-Pro with a y-turn for the preceding residue Tyr7 and either a type I/III p-turn around Pro9-IlelO or cxR orientations for these residues, which are both stabilized by an Asx turn. As determined by energy calculations, these structures were equally as stable as the kinked truns-Pro helix and could constitute key structures for analysing the conformational changes and/or the dynamics of TM VII segment induced by the ligand when interacting with the receptor.The apparent similarity in the hydropathicity profiles of G-protein-coupled receptors [ 1 -41 with that of bacteriorhodopsin [5] has led to the use of this integral membrane protein as a starting point for predicting their three-dimensional structures [6-121, even though bacteriorhodopsin is not coupled to G-proteins. The currently accepted proposal for the topography of G-protein-coupled receptors includes seven transmembrane-spanning domains (TM I-VII) joined together by extracellular and cytoplasmic loops, with the Nterminal side in the extracellular cleft and the C-terminal residues in the cytoplasm. This model, which was first deduced Correspondence to S .

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Section: Discussionmentioning

confidence: 99%

Section: Residuementioning

confidence: 99%

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Berlose

Convert

Brunissen

et al. 1994

European Journal of Biochemistry

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“…3). Val-114 (3.33) is located between Asp-113 (3.32), an amino acid essential for electrostatic interaction with the amine-group common to the ligands in the amine receptor subfamily (20), and the serine residues of the two conserved SxxxS motifs. As a result, the side chain of Val-114, is positioned to be in van der Waals contact with the bound ligand.…”

Section: Discussionmentioning

confidence: 99%

Role of group-conserved residues in the helical core of β ₂ -adrenergic receptor

Chelikani

Horn̆ák

Eilers

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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G protein-coupled receptors (GPCRs) belonging to class A contain several highly conserved (>90%) amino acids in their transmembrane helices. Results of mutational studies of these highly conserved residues suggest a common mechanism for locking GPCRs in an inactive conformation and for their subsequent activation upon ligand binding. Recently, a second set of sites in the transmembrane helices has been identified in which amino acids with small side chains, such as Gly, Ala, Ser, Thr, and Cys, are highly conserved (>90%) when considered as a group. These group-conserved residues have not been recognized as having essential structural or functional roles. To determine the role of group-conserved residues in the ␤2-adrenergic receptor (␤2-AR), amino acid replacements guided by molecular modeling were carried out at key positions in transmembrane helices H2-H4. The most significant changes in receptor expression and activity were observed upon replacement of the amino acids Ser-161 and Ser-165 in H4. Substitution at these sites by larger residues lowered the expression and activity of the receptor but did not affect specific binding to the antagonist ligand dihydroalprenolol. A second site mutation, V114A, rescued the low expression of the S165V mutant. Substitution of other group-conserved residues in H2-H4 by larger amino acids lowered receptor activity in the order Ala-128, Ala-76, Ser-120, and Ala-78. Together these data provide comprehensive analysis of group-conserved residues in a class A GPCR and allow insights into the roles of these residues in GPCR structure and function.rhodopsin ͉ G protein-coupled receptors ͉ helix packing ͉ site-directed mutagenesis ͉ molecular modeling G protein-coupled receptors (GPCRs) contain seven transmembrane helices and mediate signal transduction in response to a wide variety of extracellular stimuli. The large GPCR family is subdivided into five classes on the basis of sequence conservation. Although the ␤ 2 -adrenergic receptor (␤ 2 -AR) and the visual pigment rhodopsin are well-characterized members of class A GPCRs, rhodopsin is the only GPCR for which highresolution structural information has been obtained by protein crystallography (1, 2) and by NMR spectroscopy (3, 4). The most extensively studied ligand-activated GPCR is ␤ 2 -AR, which mediates physiological responses to adrenaline and noradrenaline and, therefore, plays a significant role in the regulation of the cardiovascular system.The class A GPCRs are made up of Ͼ1,000 unique sequences that are subdivided into several subclasses, which include the opsin, amine, peptide, and olfactory receptors. The class A receptors have been analyzed extensively in regard to structure and function with a focus on those amino acids with sequence identities of Ͼ90%, such as the conserved Asn at position 1.50 ʈ (100%) in H1, Asp at position 2.50 (93%) in H2, and the signature D/ERY and NPxxY motifs in helices H3 and H7, respectively. In addition to these polar residues, there are several conserved hydrophobic amino acids at posit...

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“…3B). Examples in β 2 AR include Asp79 (41,42), Asp113 (42,43), Cys116 (44), Asp130 (45), and Cys327 (46). Asp79 buried in the core of β 2 AR is a particularly important site, because mutations of structurally equivalent Asp residues in other GPCRs (α 2 -adrenergic, D 2 dopamine, and M1 muscarinic acetylcholine receptors) also impede receptor function (47).…”

Section: Significancementioning

confidence: 99%

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

Proc. Natl. Acad. Sci. U.S.A.

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Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light-and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligandbinding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.7-transmembrane receptor | G protein-coupled receptor | buried charge | structural bioinformatics | molecular evolution S even-transmembrane receptors (7TMRs) are present in all domains of life. In archaea and bacteria these receptors convert light energy into transmembrane ion gradients or intracellular signaling cascades. In humans 7TMRs comprise a family of more than 800 G protein-coupled receptors (GPCRs) that detect extracellular signals such as odorants, taste, light, hormones, and neurotransmitters. Although microbial and eukaryotic 7TMRs lack sequence similarity, their 3D folds share a degree of similarity that often is observed in remote evolutionary relationships identified by structure comparison algorithms (1).Recent breakthroughs in membrane protein crystallography have advanced our understanding of GPCR function by providing models of unactivated and activated receptor conformations (2-8). However, structure-based approaches for systematically quantifying differences between GPCR activation states are lacking. Here we introduce a computational approach that correlates GPCR activation with networks of electrostatic interactions in the receptor core. We show that membrane-spanning networks of ionizable residues, which we find are a common feature of microbial 7TMRs, also represent a unique signature of GPCR activation that is likely to be essential to receptor function.The molecular changes that accompany 7TMR activation were studied initially in bacteriorhodopsin and rhodopsin, the first microbial 7TMR and GPCR to be crystallized (9-11). More recently, a rapid expansion of GPCR structural information has revealed several molecular switches and structural features that are thought to be indicative of receptor activation. These include the ionic loc...

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Identification of residues required for ligand binding to the beta-adrenergic receptor.

Cited by 372 publications

References 19 publications

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Role of group-conserved residues in the helical core of β ₂ -adrenergic receptor

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

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Identification of residues required for ligand binding to the beta-adrenergic receptor.

Cited by 372 publications

References 19 publications

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Three‐Dimensional Structure of the Highly Conserved Seventh Transmembrane Domain of G‐Protein‐Coupled Receptors

Role of group-conserved residues in the helical core of β 2 -adrenergic receptor

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

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Role of group-conserved residues in the helical core of β ₂ -adrenergic receptor