Abstract:Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate … Show more
“…This is further supported by recent findings suggesting that three mutations associated with retinitis pigmentosa (a blinding disease) disrupt the ability of rhodopsin to form GPCR homomers via TM1 and TM5 [32]. …”
Section: Family a Gpcr Homomers As A Functional Unit In Mammalian Cellssupporting
G protein-coupled receptors (GPCRs) are a remarkably multifaceted family of transmembrane proteins that exert a variety of physiological effects. Although family A GPCRs are able to operate as monomers, there is increasing evidence that heteromerization represents a fundamental aspect of receptor function, trafficking and pharmacology. Most recently, it has been suggested that GPCR heteromers may play a crucial role as new molecular targets of heteromer-selective and bivalent ligands. The current review summarizes key recent developments in these topics.
“…This is further supported by recent findings suggesting that three mutations associated with retinitis pigmentosa (a blinding disease) disrupt the ability of rhodopsin to form GPCR homomers via TM1 and TM5 [32]. …”
Section: Family a Gpcr Homomers As A Functional Unit In Mammalian Cellssupporting
G protein-coupled receptors (GPCRs) are a remarkably multifaceted family of transmembrane proteins that exert a variety of physiological effects. Although family A GPCRs are able to operate as monomers, there is increasing evidence that heteromerization represents a fundamental aspect of receptor function, trafficking and pharmacology. Most recently, it has been suggested that GPCR heteromers may play a crucial role as new molecular targets of heteromer-selective and bivalent ligands. The current review summarizes key recent developments in these topics.
“…“flippant’s” capabilities are demonstrated here by reanalyzing a subset of the data in [14]. The first step is to extract the data files, stored in a ZIP archive within the package.
…”
Section: Resultsmentioning
confidence: 99%
“…PPR scaling is used with the default factor of 0.65.Fig. 1
flippant-based plotting of spectral traces underlying a subset of the data in Figure 4C–F from [14]. See text for command and details
…”
Section: Resultsmentioning
confidence: 99%
“…Based on the manual analysis of the data we conclude in [14] that the mutant rhodopsins investigated are reconstituted into proteoliposomes in monomeric form, contrary to the wild type, which is reconstituting as a homodimer. “flippant” analysis of the data shows identical trends and close matches of the results, supporting identical conclusions.…”
BackgroundThe lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps.ResultsWith the extension package “flippant” to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results.Conclusions“flippant” allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1542-y) contains supplementary material, which is available to authorized users.
“…Scramblase activity was measured using a well-established fluorescence-based assay ( Fig. 2A) (6,(17)(18)(19). Briefly, large unilamellar vesicles are reconstituted with TMEM16 and fluorescent nitrobenzoxadiazole-conjugated phosphatidylethanolamine (NBD)-PE.…”
Section: Acp-tagged Nhtmem16 Proteins Are Functionalmentioning
Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus ) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.
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