The R7‐RGS/Gβ5 protein duplex functions as a GTPase‐activating protein (GAP) to facilitate the Gαi/o intrinsic GTPase activity. The R7‐RGS subfamily of RGS (Regulator of G protein Signaling) proteins comprises RGS6, RGS7, RGS9 and RGS11. The R7 family‐binding protein (R7BP) anchors the R7‐RGS/Gβ5 duplex to the cell membrane to enhance its GAP activity. Recently it was shown that knock‐out of R7BP in mice led to decreased acute and chronic itch sensation, indicating that R7BP has a central role in itch response. We are developing interfering peptides to block the R7BP and R7‐RGS/Gβ5 interactions as potential inhibitors of chronic itch. We demonstrate here the use of cross‐linking mass spectrometry (XL‐MS) to obtain structural clues about human R7‐RGS/Gβ5/R7BP triplexes. To this end, we first transfected plasmids encoding human RGS7, Gβ5 and R7BP into Expi293 cells and purified the protein triplex to 95% purity. The triplex was treated with the cleavable cross‐linking reagent DSSO (disuccinimidyl sulfoxide) at different protein:DSSO molar ratios and analyzed by mass spectrometry. The experiment was repeated using the human RGS9/Gβ5/R7BP triplex. Both experiments yielded peptides involved in the RGS7/9‐R7BP interaction, with one R7BP peptide common to both. We believe this peptide region to be an essential interaction surface on R7BP and an ideal target for interfering peptide design to block the R7‐RGS/R7BP interaction. Taken together, we demonstrate that XL‐MS is an effective way to shed light on structural features of the R7‐RGS/Gβ5/R7BP triplex to facilitate the development of novel anti‐itch drugs.Support or Funding InformationNational Institute of Diabetes and Digestive and Kidney Diseases Intramural Funding, NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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