In the G protein-coupled receptor rhodopsin, the conserved NPxxY(x) 5,6F motif connects the transmembrane helix VII and the cytoplasmic helix 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after retinal photoisomerization. Here, we demonstrate that Ala replacement mutations within the NPxxY(x) 5,6F domain, which eliminate an interaction between aromatic residues Y306 and F313, allow formation of Meta II despite the presence of 9-demethyl-retinal. Also a disulfide bond linking residues 306 and 313 in the 9-demethylretinal-reconstituted mutant Y306C͞F313C͞C316S prevented Meta II formation, whereas the reduced form of the mutant readily transformed to Meta II after illumination. These observations suggest that the interaction between residues 306 and 313 is disrupted during the Meta I͞Meta II transition. However, this enhancement in Meta II formation is not reflected in the G protein activation, which is dramatically reduced for these mutants, suggesting that changes in the Y306 -F313 interaction also lead to a proper realigning of helix 8 after photoisomerization. The E134Q mutation, located in the second conserved motif, D(E)RY, rescues activity in 9-demethyl-retinal-reconstituted mutants to different degrees, depending on the position of the Ala replacement in the NPxxY(x) 5,6F motif, thus revealing distinct roles for the NP and Y(x) 5,6F portions. Our studies underscore the importance of the NPxxY(x) 5,6F and D(E)RY motifs in providing structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II. The dual control of the structural rearrangements secures reliable transformation of quiescent rhodopsin to activating Meta II.GPCR ͉ NPxxY motif R hodopsin is a prototypical receptor from the largest subfamily A of G protein-coupled receptors (GPCRs), which are thought to operate through similar signaling mechanisms (1, 2). The only known crystal structure of a GPCR, that of rhodopsin (3), confirmed the presence of seven transmembrane helices (H). The structure describes the receptor in its inactive ground state with the bound inverse agonist, 11-cis-retinal. The -amino group of K296 in H-VII tethers the chromophore 11-cis-retinal to opsin via a covalent Schiff base (SB) bond (Fig. 1). A salt-bridge between the protonated SB and the counterion, the conserved Glu-113 in H-III, contributes largely to constrain the receptor in the inactive conformation. Additional constraints involve hydrogen bond networks and hydrophobic interactions linking transmembrane helices, sequestration of the Arg residue of the D(E)RY motif in the hydrophobic milieu, and intramolecular interactions within the NPxxY(x) 5,6 F region (3, 4). Absorption of light energy isomerizes the 11-cis-retinylidene chromophore, generating the agonist all-trans-retinylidene in situ. Subsequent thermal relaxation of the retinal-protein complex leads within milliseconds to an e...

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanismbased pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.