Mechanism for adhesion G protein-coupled receptor GPR56-mediated RhoA activation induced by collagen III stimulation

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.

Figure 1. Cell surface expression of GPR56 and its mutant protein.

(A). Schematic representation of GPR56 protein with three GPR56^C mutations indicated. (B). Detection of membrane expressed GPR56 by biotinylation experiments. Cells transfected with wild type Gpr56 and L640R mutant cDNA have comparable cell surface expression of GPR56^C, whereas cells transfected with either E496K or R565W show much reduced or absent cell surface expression of GPR56^C. (C). Bar graphs depict optical density of the western blot of GPR56^C in (B). (D) Analysis of the N- and C- terminal fragment association on plasma membrane by coimmunoprecipitation (IP) experiments. The association of GPR56^N and GPR56^C was observed in both wild type and L640R mutant receptors. (E). Detection of colocalization of GPR56^N and GPR56^C on cell plasma membrane by immunostaining. Scale bar: 10 µm.

Figure 2. Collagen III induces a shift of GPR56^C from the non-raft to raft fractions.

(A). Western blot analysis of the lipid raft fractionation of 293T cells transfected with wild type Gpr56 using anti-GPR56^C (199), and GPR56^N (H11) to detect the C-terminal and N-terminal of GPR56, respectively. Cholera toxin B subunit (CTB), which binds to ganglioside GM1, served as a marker for lipid raft. Different lane numbers correspond to different fractions after sucrose gradient centrifugation. Higher protein bands in fraction 9–11 likely represent protein aggregate. Arrowhead indicating the corresponding GPR56^C and arrow showing the responding GPR56^N. (B). The relative optical intensity of GPR56^C observed in A was measured using Image QuantTL program and presented as mean ± SE in a linear plot. n = 3, *P = 0.0124, Student t test. (C) Western blot analysis of the lipid raft fractionation of 293T cells transfected with L640R mutant cDNA. Higher protein bands in fraction 8–11 likely represent protein aggregate. Arrowhead indicating the corresponding GPR56^C and arrow showing the responding GPR56^N. (D). The relative optical intensity of GPR56^C observed in C was measured using Image QuantTL program and presented as mean ± SE in a linear plot. n = 3, *P = 0.006, Student t test.

Figure 3. Collagen III treatment causes a reduced plasma membrane associated GPR56^N.

(A–L). Immunostaining of GPR56^N and GPR56^C. Collagen III stimulation results in a decreased level of GPR56^N. (M). Detection of surface expressed GPR56^N by flow cytometry. Surfaced expressed GPR56^N was probed with anti- GPR56^N (CG4) antibody, followed by flow cytometry analysis. Shown are representative histograms. (N) Bar graphs show the mean and SD of the geometric mean fluorescence intensity (MFI) of GPR56^N expression. n = 3, *P = 0.02, **P = 0.008. (O) Western blot detection of GPR56^N in the cell conditioned media with or without collagen III treatment. Collagen III treatment results in a higher GPR56^N in the cell conditioned media.

Figure 4. L640R mutation attenuates RhoA activation upon collagen III stimulation.

(A) The addition of collagen III caused an increased level of GTP-RhoA in cells transfected with wild type GPR56, but not in cells transfected with L640R mutant. Total RhoA expression in the cell lysate served as a loading control. (B) Bar graph of the relative optical density of GTP-RhoA. n = 3, *P = 0.024. (C) The addition of Calpeptin resulted in a comparable elevation of GTP-RhoA in cells transfected either wild type or L640R mutant Gpr56 cDNA.

Figure 5. Position of L640 in the GPR56 transmembrane helices.

(A) L640 is positioned on the transmembrane helix 7 close to the extracellular side. Its side chain faces the extracellular cavity where ligands typically bind to in other receptor families. L640 is colored magenta. (B) The mutation of Leucine to Arginine creates a long charged side chain that may reside in multiple conformations. The side chain of Arginine (white and blue) is able to reach residues from other transmembrane helices and possibly be involved in new interactions that the Leucine side chain is unable to. These interactions may favor a locked inactive conformation of the receptor. The GPR56 model was made by MODELLER based on the secretin family structure (PDB ID: 4L6R). Nitrogen and oxygen atoms are colored blue and red, respectively. Figure was drawn by PYMOL. (C) Alignment of the TM7 in GPR56 orthologs. ClustalW was used to perform an amino acid sequence alignment of the TM7 in GPR56 orthologs. L640 is highly conserved among GPR56 orthologs.

Figure 6. Alignment of TM7 in adhesion-GPCRs.

ClustalW was used to perform an amino acid sequence alignment of the L640 residue in adhesion-GPCRs. L640 is not conserved among the majority of adhesion GPCRs.

Figure 7. The signaling of GPR56.

The binding of collagen III with wild type GPR56 (dark blue) releases GPR56^N from the membrane-bound GPR56^C and triggers the association of GPR56^C with lipid rafts, thus activating its downstream signaling molecular RhoA. For the L640R mutant (light blue), the binding of collagen III to the receptor fails to couple to Gα_12/13 and activate RhoA, despite its ability to release GPR56^N from the membrane-bound GPR56^C and to trigger the association of GPR56^C with lipid rafts.