Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors.
Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.
Knierim
Bernhard
B
Institut für Medizinische Physik und Biophysik (CCM), Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Hofmann
Klaus Peter
KP
Gärtner
Wolfgang
W
Hubbell
Wayne L
WL
Ernst
Oliver P
OP
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry; D012243:Rhodopsin / Q000819:agonists* / Q000737:chemistry*; D012545:Schiff Bases; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels
C110176:9-demethylretinal; D011522:Protons; D012545:Schiff Bases; D013113:Spin Labels; D012243:Rhodopsin; D012172:Retinaldehyde