Allosteric coupling from G protein to the agonist-binding pocket in GPCRs (original) (raw)
Related papers
2001
G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the  2 adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the  2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.
Agonist-bound structures of G protein-coupled receptors
Current Opinion in Structural Biology, 2012
G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the b 2adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.
Structural Basis for Activation of G-Protein-Coupled Receptors
Pharmacology and Toxicology, 2002
Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the b 2 -adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where disruption of intramolecular interactions between TM3 and TM6 leads to a major conformational change of TM6 relative to the rest of the receptor. This conclusion was based on experiments in which environmentally sensitive, sulfhydryl-reactive fluorophores were site-selectively incorporated into wild-type and mutant b 2 -adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the b 2 -adrenergic receptor in Xenopus Laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co-expression with the cAMP sensitive Cl ª channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.
Structure of a β1-adrenergic G-protein-coupled receptor
Nature, 2008
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 b 1 -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 a-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 b 1 -adrenergic receptor and binding of carazolol to the b 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the b 2 -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.
Structure of a beta1-adrenergic G-protein-coupled receptor
Nature, 2008
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 b 1 -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 a-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 b 1 -adrenergic receptor and binding of carazolol to the b 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the b 2 -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.
Journal of Biological Chemistry, 2001
To investigate their role in receptor coupling to G q , we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣ 1badrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the ␣ 1b -AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg 254 and Lys 258 in the third intracellular loop and Lys 291 at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val 147 and Leu 151 in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg 254 , Lys 258 , as well as Leu 151 are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the ␣ 1b -AR. These findings contribute to the delineation of the molecular determinants of the ␣ 1b -AR/G q interface.
Proceedings of the National Academy of Sciences, 2015
Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive ...
Structural basis of G protein-coupled receptor function
Molecular and Cellular Endocrinology, 1999
The vast majority of extracellular signaling molecules, like hormones and neurotransmitters, interact with a class of membranous receptors characterized by a uniform molecular architecture of seven transmembrane a-helices linked by extra-and intracelluar peptide loops. In a reversible manner, binding of diverse agonists to heptahelical receptors leads to activation of a limited repertoire of heterotrimeric guanine nucleotide-binding proteins (G proteins) forwarding the signal to intracellular effectors such as enzymes and ion channels. Proper functioning of a G protein-coupled receptor is based on a complex interplay of structural determinants which are ultimately responsible for receptor folding, trafficking and transmembrane signaling. Applying novel biochemical and molecular biological methods interesting insights into receptor structure/function relationships became available. These studies have a significant impact on our understanding of the molecular basis of human diseases and may eventually lead to novel therapeutic strategies. : S 0 3 0 3 -7 2 0 7 ( 9 9 ) 0 0 0 1 7 -9
Crystal structure of the human β2 adrenergic G-protein-coupled receptor
Nature, 2007
Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human b 2 adrenoceptor (b 2 AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å / 3.7 Å resolution. The cytoplasmic ends of the b 2 AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the b 2 AR are not seen. The b 2 AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (T M)3 and T M 6, involving the conserved E / DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the b 2 AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.