FIGURE 2.

Identifying protein interactions that mediate nAChR trafficking, localization, and signaling. (A) Cell surface receptors can be selectively labeled with an anti-α7 nAChR monoclonal Ab. Cell surface labeling (at 4°C) can be combined with DSP in order to crosslink the receptor interactome. Alternatively, antibody labeling and crosslinking can be used to examine changes in the receptor interactome between internalized and cell surface nAChRs. (B) Chemical crosslinking can be used to study the dynamics of nAChR–protein interactions under various ligand treatment conditions. (C) Experimental evidence on α7 nAChR interactions with GAP-43 and CaM in developing neural cells. BS₃ was used to crosslink the α7 nAChR interactome from differentiating cells. An IP was utilized to purify the receptor, which was visualized by SDS-PAGE. Protein identity was confirmed using LC-ESI MS and Western blot. These experiments demonstrate dynamic changes in CaM/GAP-43 association with α7 nAChR in response to nicotine activation (Nordman and Kabbani, 2012).