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...

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Background: Age-related arterial alterations affecting cells, matrix and biomolecules are the main culprit for initiation and progression of cardiovascular disease. The objective of this study is to gain further insights into the complex mechanism of elastic tissue ageing in human aortic blood vessels. Methods: One hundred and nineteen human aortic tissue samples were collected from adult patients (101 males, 18 females; age 40–86 years) undergoing coronary artery bypass grafting. Overall extracellular matrix architecture was examined by multiphoton laser scanning microscopy and histology. Matrix metalloproteinases 2 and 9, corresponding tissue inhibitors 1 and 2 as well as desmosine were determined. mRNA levels of tropoelastin were assessed by quantitative RT-PCR. Results: Age-related destruction of the vascular elastic laminas as well as a loss of interlamina cross-links were observed by laser scanning microscopy. These results were confirmed by histology indicating increasing interlamina gaps. There were no significant differences in matrix turnover or desmosine content. A steady decrease in tropoelastin mRNA by about 50% per 10 years of age increase was observed. Conclusions: Our findings indicate that ageing is accompanied by a destruction of the elastic vascular structure. However, tropoelastin expression analysis suggests that elastogenesis occurs throughout life with constantly decreasing levels.