Coupling ligand structure to specific conformational switches in the β2-adrenoceptor

G protein–coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human β2-adrenoceptor (β2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state1,2,3. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the β2-AR.

[1]  P. Corvol,et al.  Lessons from constitutively active mutants of G protein-coupled receptors , 2002, Trends in Endocrinology & Metabolism.

[2]  J. Ballesteros,et al.  Activation of the β2-Adrenergic Receptor Involves Disruption of an Ionic Lock between the Cytoplasmic Ends of Transmembrane Segments 3 and 6* , 2001, The Journal of Biological Chemistry.

[3]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[4]  T. Mielke,et al.  Electron crystallography reveals the structure of metarhodopsin I , 2004, The EMBO journal.

[5]  H Weinstein,et al.  Agonists induce conformational changes in transmembrane domains III and VI of the β2 adrenoceptor , 1997, The EMBO journal.

[6]  Mark H Ellisman,et al.  A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells , 2005, Nature Methods.

[7]  J. Ballesteros,et al.  Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. , 2002, The Journal of biological chemistry.

[8]  L. Pardo,et al.  Linking agonist binding to histamine H1 receptor activation , 2005, Nature chemical biology.

[9]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[10]  Bryan L Roth,et al.  Evidence for a Model of Agonist-induced Activation of 5-Hydroxytryptamine 2A Serotonin Receptors That Involves the Disruption of a Strong Ionic Interaction between Helices 3 and 6* 210 , 2002, The Journal of Biological Chemistry.

[11]  H. Mchaourab,et al.  Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence. , 2002, Biochemistry.

[12]  B. Kobilka.  Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. , 1995, Analytical biochemistry.

[13]  J. Ballesteros,et al.  [19] Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors , 1995 .

[14]  Xavier Deupi,et al.  Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists* , 2005, Journal of Biological Chemistry.

[15]  M. Lohse,et al.  Molecular basis of inverse agonism in a G protein–coupled receptor , 2005, Nature chemical biology.

[16]  Junmei Wang,et al.  Development and testing of a general amber force field , 2004, J. Comput. Chem..

[17]  Yang Xiang,et al.  Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states. , 2004, The Journal of biological chemistry.

[18]  P Ghanouni,et al.  Functionally Different Agonists Induce Distinct Conformations in the G Protein Coupling Domain of the β2Adrenergic Receptor* , 2001, The Journal of Biological Chemistry.

[19]  H. Khorana,et al.  Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin , 1996, Science.

[20]  Francesca Fanelli,et al.  Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation. , 2002, Molecular pharmacology.

[21]  Manfred Burghammer,et al.  Structure of bovine rhodopsin in a trigonal crystal form. , 2003, Journal of molecular biology.

[22]  S. Green,et al.  A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. , 1993, The Journal of biological chemistry.