Abstract
The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), and 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6. The resulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evidence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions 271(6.33) and 272(6.34). The data suggest that activation of G protein-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside. Based on these preferred conformations, it is suggested that the spectral changes reflect an agonist-promoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer.
Highlights
The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the 2 adrenergic receptor, a prototype hormone-activated G protein-coupled receptor
Cysteine Substitutions at the Cytoplasmic Side of transmembrane segment (TM) 6 —To achieve the possibility of site-selective, covalent incorporation of the sulfhydryl-reactive fluorophore IANBD into the purified 2AR, we wanted to reduce the number of endogenous cysteines available for chemical derivatization
We have previously shown that agonist binding to the purified and IANBD-labeled wild type 2AR is accompanied by a reversible decrease in fluorescence intensity and that the spectral changes reflect conformational changes involved in receptor activation (9)
Summary
The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the 2 adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside Based on these preferred conformations, it is suggested that the spectral changes reflect an agonistpromoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer. Employment of a computational method, which incorporated the complex hydrophobic/hydrophilic environment in which the inserted cysteines reside, allowed a prediction of the preferred conformations of the attached IANBD moiety Analysis of these preferred conformations in context of a receptor model supported the possibility that TM 6 undergoes movements that are similar to those predicted to take place in rhodopsin during photo-activation
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