TY - JOUR

T1 - Structural basis for activation of G-protein-coupled receptors.

AU - Gether, Ulrik

AU - Asmar, Fazila

AU - Meinild, Anne Kristine

AU - Rasmussen, Søren G F

N1 - Keywords: Animals; Cystic Fibrosis Transmembrane Conductance Regulator; GTP-Binding Proteins; Molecular Conformation; Receptors, Adrenergic, beta-2; Receptors, Cell Surface; Structure-Activity Relationship; Xenopus laevis

PY - 2002

Y1 - 2002

N2 - 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 beta2-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 beta2-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 beta2-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- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.

AB - 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 beta2-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 beta2-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 beta2-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- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements.

U2 - 10.1034/j.1600-0773.2002.910607.x

DO - 10.1034/j.1600-0773.2002.910607.x

M3 - Journal article

C2 - 12688373

VL - 91

SP - 304

EP - 312

JO - Basic and Clinical Pharmacology and Toxicology

JF - Basic and Clinical Pharmacology and Toxicology

SN - 1742-7835

IS - 6

ER -