Instead of a relatively continuous and homogenous fluid of amphipathic lipids interspersed with a mosaic of proteins, it has been found that the plasma membrane contains nanoscale domains of sphingolipids, cholesterol, and membrane proteins, which together form what is referred to as ‘lipid rafts’ that function as receptor signaling platforms. Lipid rafts are resistant to cold nonionic detergent treatment, causing them to float to the top fraction of isopycnic sucrose gradients; thus they are named detergent resistant membranes. Proteins that associate with lipid rafts are defined as those that co-fractionate with DRM fractions. Therefore, cold-detergent extraction and membrane fractionation have been Salvianolic-acid-B extensively used to identify proteins associated with lipid rafts. It was recently reported that a low level of Pimozide GPR56C is constitutively associated with membrane lipid rafts. However, it is not known whether there is a dynamic presence of GPR56 in the lipid raft upon ligand stimulation. We set out to test the hypothesis that lipid raft association is required for GPR56 signaling. HEK 293T cells transfected with GPR56 cDNA were stimulated with either collagen III or acetic acid for 5 minutes. The cells were lysed in the presence of detergenton ice and subjected to DRM fractionation. GPR56N is tethered non-covalently with GPR56C on the plasma membrane, and therefore is restrictedly present in the non-raft fractions. Consistent with a previous report, we did detect a low basal level of GPR56C in the lipid raft fractions. Interestingly, we observed a significant shift of GPR56C from nonraft to lipid raft fractions upon collagen III stimulation, indicating that GPR56 probably needs lipid rafts as a platform for its signal transduction. DRM analysis was performed using this mutant receptor. Our result showed that the mutant GPR56C also translocated to lipid raft fractions after ligand stimulation, similar to the behavior of wild type GPR56. This data indicated that this disease-associated Cterminal mutation does not disrupt collagen III-induced association of GPR56 with plasma membrane lipid nanodomains. Thus, collagen III binds both wild type and the L640R mutant, resulting in the C-terminal fragment associating with DRMs. We previously showed that Collagen III is a ligand of GPR56 in the developing brain. Upon binding to collagen III, GPR56 activates RhoA via coupling to Ga12/13. Here, we discover that collagen III binding also induces release of the GPR56N fragment, allowing the GPR56C fragment to associate with DRMs. Surprisingly, the L640R mutation does not inhibit these processes, but instead blocks downstream RhoA activation. Like most other adhesion GPCRs, GPR56 is autocatalytically cleaved through the GPS motif between amino acids histidine-381 and leucine-382 into N- and C-terminal fragments, GPR56N and GPR56C, respectively. Although mutations in the GPS domain disrupt this cleavage 
and cause human BFPP disease, the biological significance of this cleavage is not entirely clear. We previously showed that the cleaved GPR56N remains associated with GPR56C at the plasma membrane. Furthermore, work from Hall’s group showed that overexpression of GPR56C alone results in constitutive activation of RhoA. We therefore hypothesized that the association of GPR56N and GPR56C keeps the receptor in an inactivated state, and the binding of collagen III activates the receptor by removing GPR56N from GPR56C.