Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.
Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.
Freie Universität Berlin, Institut für Experimentalphysik, Arnimallee 14, D-14195 Berlin, Germany.
Kristina
K
Kirchberg
Tai-Yang
TY
Kim
Martina
M
Möller
Darko
D
Skegro
Gayathri
G
Dasara Raju
Joachim
J
Granzin
Georg
G
Büldt
Ramona
R
Schlesinger
Ulrike
U
Alexiev
D000818:Animals; D016880:Anisotropy; D019393:Arrestin / Q000737:chemistry*; D002417:Cattle; D018360:Crystallography, X-Ray / Q000379:methods; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008968:Molecular Conformation; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry*; D015398:Signal Transduction; D013053:Spectrophotometry / Q000379:methods
D019393:Arrestin; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin