There are a total of 33 members in the family in both humans and mice, present in almost every organ system with physiological functions in development, reproduction, immunity, neuronal and epithelial function, as well as tumorigenesis. Structurally, they are differentiated from other subgroups of GPCRs by the presence of an exceptionally long extracellular N-terminal region and juxtamembrane GPCR autoproteolysis-inducingdomain. Most members of the adhesion GPCRs undergo GAIN domain-mediated autoproteolytic process at the GPCR proteolysis sitemotif to produce an N-terminal fragment and a Cterminal fragment. The biological significance of this autocleavage and its implication in receptor signaling remain largely unknown. GPR56 is one important member of the adhesion GPCR family, as mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria. Additionally, GPR56 has also been reported to play a critical role in cancer progression by regulating angiogenesis. We recently discovered that collagen III is a ligand of GPR56 in Adhesion G protein-coupled receptorsare a family of noncanonical seven transmembrane spanningreceptors. There are a total of 33 members in the family in both humans and mice, present in almost every organ system with physiological functions in development, reproduction, immunity, neuronal and epithelial function, as well as tumorigenesis. Structurally, they are differentiated from other subgroups of GPCRs by the presence of an exceptionally long extracellular N-terminal region and juxtamembrane GPCR autoproteolysis-inducingdomain. Most members of the adhesion GPCRs undergo GAIN domain-mediated autoproteolytic process at the GPCR proteolysis sitemotif to produce 
an N-terminal fragment and a Cterminal fragment. The biological significance of this autocleavage and its implication in receptor signaling remain largely unknown. GPR56 is one important member of the adhesion GPCR family, as mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria. Additionally, GPR56 has also been reported to play a critical role in cancer progression by regulating angiogenesis. We recently discovered that collagen III is a ligand of GPR56 in the developing brain and that the binding of GPR56 to collagen III activates RhoA by coupling to Ga12/13. In the context of cancer biology, GPR56 was shown to bind tissue transglutaminase. Although it is unclear whether the binding of TG2 to GPR56 triggers downstream signaling, deleting the binding site of TG2 in GPR56 activates PKCa and elevates VEGF production in a melanoma cell line MC-1. Nevertheless, the molecular mechanism underlying GPR56 signaling, including the importance of GPR56N-GPR56C interactions, remain poorly understood. To gain insight into GPR56 signaling, we explored the molecular mechanism of the activation of GPR56 signaling by collagen III using wild type GPR56 and its BFPP Eleutheroside-E associated mutants. Our results demonstrate that collagen III binding causes the release of Sarafloxacin HCl GPR56N from cell surfaces and induces GPR56C redistribution to detergent resistant membrane fragments, the biochemical correlate of lipid rafts. Furthermore, L640 is an evolutionarily conserved amino acid in GPR56 across multiple species, and a BFPP-associated mutation at this amino acid residue, L640R, specifically abolishes collagen III-induced RhoA activation.