Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1

Corin, Karolina; Philipp, B.; Geißler, Sven; Wienken, Christoph J.; Duhr, Stefan; Braun, Dieter; Zhang, Shuguang

doi:10.1038/srep00172

Cited by 43 publications

(43 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This strategy has 13 allowed efficient expression and purification of the receptors -for characterization purposes-without changing, in principle, the native protein expression and cellular localization. Our preliminary purification efforts resulted in aggregated receptors that did not show functional activity (data not shown), we solved this problem by purifying the receptors in the presence of their ligand, that had been successfully used in the purification of other receptors [34,35]. In this case we were able to obtain purified receptors that did not appear to be aggregated judged from the UV spectra and electrophoretic pattern.…”

Section: Discussionmentioning

confidence: 70%

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

et al. 2015

View full text Add to dashboard Cite

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G-protein-coupled receptors superfamily and they have been described to heterodimerize triggering an anomalous physiological state that would underly depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody, and employed ligand assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology, but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with FRET measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modelling suggests that there is a coincidence between zinc binding sites and heterodimerization interfaces for the serotonin receptor.Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

show abstract

Section: Discussionmentioning

confidence: 70%

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

et al. 2015

View full text Add to dashboard Cite

show abstract

“…[13][14][15] The T-Rex system was used to make the T4L variants in the same manner as the native receptors. Final induction conditions for hOR17-4T4L were 1 mg ml À1 of tetracycline with 5 mM sodium butyrate, and for hVN1R1 were 1 mg ml À1 of tetracycline with 1 mM sodium butyrate.…”

Section: Resultsmentioning

confidence: 99%

“…View Article Online determined, 13,14 new detergent screens were performed for hOR17-4T4L and hVN1R1-T4L.…”

Section: Systematic Detergent Screening For Receptor Solubilizationmentioning

confidence: 99%

“…13,14 When the appropriate density was reached, cells were induced with tetracycline and sodium butyrate. After two days, cells were scrape harvested.…”

Section: Cell Extract Preparationmentioning

confidence: 99%

“…[13][14][15] Briefly, frozen cell pellets were thawed on ice. Cells were resuspended in PBS containing protease inhibitors.…”

Section: Receptor Purificationmentioning

confidence: 99%

See 2 more Smart Citations

Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs

et al. 2012

Self Cite

View full text Add to dashboard Cite

The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactoryrelated receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (M W = 178.23) and myrtenal (M W = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies.

show abstract

Discovery of the first self‐assembling peptide, study of peptide dynamic behaviors, and G protein‐coupled receptors using an Aviv circular dichroism spectropolarimeter

Zhang

2018

Biopolymers

Self Cite

View full text Add to dashboard Cite

Circular dichroism (CD) spectroscopy is a useful technique to study the structure and dynamics of peptides, proteins and nucleic acids. CD is particularly useful because sample volumes may be as low as 50 μL, it provides high precision and sensitivity, and it achieves a good signal to noise ratio. CD characterizes molecular conformational changes in real time by finely controlling temperature, pH, and titrating urea and guanidine·HCl which is necessary for studying protein folding. Although CD does not provide detailed structure at the atomic level, it provides a global structural framework. Researchers use CD to observe molecular phenomena, namely how macromolecules unfold/refold and their overall self‐assembly/disassembly. Using CD to monitor a peptide structure, I serendipitously discovered the self‐assembling peptide EAK16 from yeast protein Zuotin. This unusual peptide formed a new type of nanofiber scaffold hydrogel material. The discovery in 1990 opened a new field in the design and study of numerous self‐assembling peptides, thereby launching the area of peptide nanobiotechnology. In this review, I reflect on my personal discoveries of several self‐assembling peptides, investigations into the dynamic behaviors of peptides, as well as the impact of the work on society. I also describe studies of natural membrane proteins and engineered membrane proteins using CD. Furthermore, I enjoyed numerous and close interactions with Jack Aviv since 1997. He generously supported 10 high impact workshops (Crete and Mikonos) and meetings in various countries around the world that left fond memories of many young researches who later became leading scientists in their respective fields.

show abstract

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1

Cited by 43 publications

References 36 publications

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization

Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs

Discovery of the first self‐assembling peptide, study of peptide dynamic behaviors, and G protein‐coupled receptors using an Aviv circular dichroism spectropolarimeter

Contact Info

Product

Resources

About