G protein-coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical development and lamination

Abstract

GPR56, an orphan G protein-coupled receptor (GPCR) from the family of adhesion GPCRs, plays an indispensable role in cortical development and lamination. Mutations in the GPR56 gene cause a malformed cerebral cortex in both humans and mice that resembles cobblestone lissencephaly, which is characterized by overmigration of neurons beyond the pial basement membrane. However, the molecular mechanisms through which GPR56 regulates cortical development remain elusive due to the unknown status of its ligand. Here we identify collagen, type III, alpha-1 (gene symbol Col3a1) as the ligand of GPR56 through an in vitro biotinylation/proteomics approach. Further studies demonstrated that Col3a1 null mutant mice exhibit overmigration of neurons beyond the pial basement membrane and a cobblestone-like cortical malformation similar to the phenotype seen in Gpr56 null mutant mice. Functional studies suggest that the interaction of collagen III with its receptor GPR56 inhibits neural migration in vitro. As for intracellular signaling, GPR56 couples to the Gα(12/13) family of G proteins and activates RhoA pathway upon ligand binding. Thus, collagen III regulates the proper lamination of the cerebral cortex by acting as the major ligand of GPR56 in the developing brain.

Fig. 1.

Identification of GPR56 ligand. (A–D) GPR56^N-hFc binds specifically the putative ligand of GPR56 (green) in meninges and pial BM (A) and MFs (C). GPR56^Ndel-hFc was used as a negative control (B and D). A and B are composite images combined into a single image. Nuclear counterstain was performed by Hoechst 33342 (blue). (Scale bars: A and B, 200 μm; C and D, 100 μm.) (E) SDS/PAGE analysis of purified GPR56 ligand(s) eluted from streptavidin affinity resin. The putative ligand of GPR56 is a large protein with a molecular weight of 160–260 kDa (arrowheads). (F) Summary of MS results from two independent purifications. Total peptides sequenced and proteins specifically identified by GPR56^N are indicated. Collagen III appeared as the top candidate based on the high number of matched peptides. (G) The association of collagen III and GPR56^N was confirmed by co-IP. Collagen III was specifically pulled down by GPR56^N, but not GPR56^Ndel. Conversely, only GPR56^N was pulled down by anti-collagen III antibody, but not GPR56^Ndel.

Fig. 2.

Collagen III is the major binding partner of GPR56 in the mouse developing brain. (A) Collagen III immunostaining in E12.5 developing cortex. (A′) Higher-magnification views of the boxed region. (B–G) Double IHC of collagen III/PECAM and collagen III/Zic on E11.5 wild-type mouse brains. (H–J) Double IHC of collagen III (green) and collagen IV (red) in E12.5 neocortex. (K and L) GPR56^N-hFc (green) detected a strong signal in the meninges and pial BM of E12.5 Col3a1^+/+ brain but failed to detect any signals in the Col3a1^−/− brain. Nuclear counterstain was performed by Hoechst 33342 (blue). (M and N) Abundant GPR56^N-hFc–specific (green) binding was detected in primary MFs derived from the meninges of Col3a1^+/+ but not Col3a1^−/− mice. Nuclear counterstain was performed by Hoechst 33342 (blue). (Scale bars: A and K–N, 100 μm; A′, 50 μm; B–J, 10 μm.)

Fig. 3.

Deletion of the Col3a1 gene results in neuronal ectopias and abnormal cortical lamination. (A and B) Nissl staining of E18.5 Col3a1^+/+ and Col3a1^−/− brains. Neuronal ectopias (arrowheads) were seen in Col3a1^−/− mouse brain (B). (C–H) Immunostaining of Cux1 (red in C–E), Tbr1 (red in F–H), and CTIP2 (green in F–H) in E18.5 Col3a1^+/+, Col3a1^−/−, and Gpr56^−/− brains. Neurons positive for Cux1, Tbr1, and CTIP2 were detected in the ectopias of Col3a1^−/− and Gpr56^−/− brains (D, E, G, and H). (Scale bars: A and B, 500 μm; C–H, 100 μm.)

Fig. 4.

Collagen III inhibits neuronal migration. (A–E) NPC migration assay. In contrast to the robust NPC migration observed in the control media, recombinant collagen III inhibited NPC migration from Gpr56^+/− neurospheres (C), but not from Gpr56^−/− neurospheres (D). The addition of exogenous GPR56^N significantly blocked collagen III-mediated migration inhibition (E). (F) The degree of collagen III-mediated migration inhibition was quantified as a percentage of the migrating neurospheres. Data are presented as mean ± SD; n = 7 for all groups except for the competition experiment, where n = 6. *P = 0.0032, **P = 0.00046, Student t test. (G–H) Double IHC of Nestin and Tuj1 of the cultured neurospheres. (G′–H′) Higher-magnification views of the boxed regions in G and H. (Scale bars: A–E, G, and H, 100 μm; G′–H′, 25 μm.)

Fig. 5.

Migrating neurons express GPR56. (A–I) Immunostaining of Tuj1 (red) and GPR56 (green) in E12.5 (A–C), E14.5 (D–F) neocortex, and 2-d in vitro-cultured (2DIV) primary cortical neurons (G–I). GPR56 was expressed in the Tuj1⁺ cell population. (J–L) Double IHC of collagen III/GPR56 in E12.5 neocortex. (M and N) Double IHC of collagen III/Tuj1 and collagen III/Nestin in E12.5 neocortex. (O) Triple IHC of collagen IV, Nestin, and Tuj1 in E11.5 neocortex. (P) Immunoelectron microscopy of collagen III. Immunogold-positive collagen III (arrowheads) is expressed in the pial BM. Both migrating neuron (MN) and radial glial endfeet (EF) have direct contact to the collagen III-containing pial BM. (Scale bars: A–F, 50 μm; G–I, 25 μm; J–O, 10 μm; P, 500 nm.)

Fig. 6.

The interaction of GPR56 and its ligand collagen III activates RhoA by coupling to Gα_12/13. (A) RhoA activation in NIH 3T3 cells. The addition of collagen III caused an increased level of GTP-RhoA, which was dependent on the presence of GPR56 and Gα₁₃. (B) Collagen III induced the elevation of GTP-RhoA level in NPCs derived from Gpr56^+/− but not Gpr56^−/− brains. Total RhoA expression in the cell lysate served as a loading control.