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Abstract
GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family.
Highlights
Adhesion G protein-coupled receptors (GPCRs) are a family of noncanonical seven transmembrane spanning (7TM) receptors
Our results demonstrate that collagen III binding causes the release of GPR56N from cell surfaces and induces GPR56C redistribution to detergent resistant membrane fragments (DRMs), the biochemical correlate of lipid rafts
PER Eukaryotic Membrane Protein Extraction Reagent Kit was from Thermo Scientific; Streptavidin–agarose beads from Sigma; RIPA buffer from Boston Bioproducts; Protease inhibitor cocktail (EDTA-free) from Roche Diagnostics; Collagen III protein was from AbCam; Mouse GPR56 cDNA cloned into pCDNA3.1(+) vector as described previously [11]; GPR56 mutations were created by site-directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene), as previously described [12]; Cholera toxin B subunit (CTB)–Alexa Fluor 488 were purchased from Invitrogen
Summary
Adhesion G protein-coupled receptors (GPCRs) are a family of noncanonical seven transmembrane spanning (7TM) receptors. 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 [1]. They are differentiated from other subgroups of GPCRs by the presence of an exceptionally long extracellular N-terminal region and juxtamembrane GPCR autoproteolysis-inducing (GAIN) domain [2,3,4]. The molecular mechanism(s) underlying GPR56 signaling, including the importance of GPR56N-GPR56C interactions, remain poorly understood
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