Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies.
The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.
Granier
Sébastien
S
Department of Molecular, Stanford University School of Medicine, Stanford, California 94305, USA.
Kim
Samuel
S
Shafer
Aaron M
AM
Ratnala
Venkata R P
VR
Fung
Juan José
JJ
Zare
Richard N
RN
Kobilka
Brian
B
D000319:Adrenergic beta-Antagonists / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D019393:Arrestin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D011412:Propanolamines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship
D000319:Adrenergic beta-Antagonists; D019393:Arrestin; D008024:Ligands; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; C026777:ICI 118551; D003545:Cysteine