Figure 8.
Figure 8. Plectin and GPR56 physically and genetically interact in peripheral nerve. (A) GTP-RhoA assay to assess Gpr56 signaling in response to exogenous ligands. Addition of collagen III, a known activating ligand of Gpr56, to HEK 293T cells coexpressing Gpr56 result in elevation of GTP-RhoA levels over control (untransfected). Addition of prion protein (FT) does not induce Gpr56 signaling through RhoA. (B) Gpr56 Presto-Tango luciferase assay to assess Gpr56 signaling in HTLA cells. Addition of collagen III results in a significant elevation of luciferase compared with AcOH alone control (*, P < 0.05, unpaired Student’s t test). Addition of FT did not result in a significant elevation of luciferase relative to PBS controls (P = 0.0647, unpaired Student’s t test). (C) cAMP ELISA assay to assess cAMP signaling through Gpr56 and Gpr126 (positive control) after addition of FT. Addition of FT to HEK 293T cells expressing Gpr126 results in a significant elevation of cAMP (**, P < 0.01, unpaired Student’s t test) compared with PBS controls. Addition of FT to cells expressing Gpr56 does not induce cAMP signaling (P = 0.4575, unpaired Student’s t test). (A–C) Data are representative of three biologically independent experiments. Error bars represent SEM. (D) Co-IP to assess interactions between GPR56 and plectin in P5 WT SNs (n = 8 nerves from four animals per replicate; n = 3 replicates). Lane 1: plectin coimmunoprecipitates with GPR56 when the NTF of GPR56 fused to human IgG Fc (NTF-hFc) is used as a bait but not when pulling down with hFc alone (lane 2). (E) Analysis of plectin levels in P6 SNs. Plectin is significantly increased in Gpr56−/− nerves over sibling controls (**, P < 0.002, unpaired Student’s t test, n = 12 nerves from six animals; error bars represent SEM). (F) Graphic model: Gpr56 promotes function of developing and mature SCs. During early SC development, Gpr56 signals through Gα12/13 and RhoA to modulate the actin cytoskeleton for efficient radial sorting of axons. During SC maturation/homeostasis, Gpr56 functions upstream of SREBP1 to regulate myelin thickness and Remak SC biology, potentially through SRE signaling downstream of RhoA (gray dashed arrow). Concordantly, Gpr56 regulates mature myelin domain organization and myelin maintenance through direct interactions with the cytolinker protein plectin, which has been shown to physically interact with additional regulators of myelin domain organization (utrophin and β-dystroglycan). We therefore hypothesize that plectin serves as a docking center for cell signaling molecules that govern the partitioning of myelin into mature subdomains (Cajal bands and appositions) and helps generate these structures through its interactions with the cytoskeleton.

Plectin and GPR56 physically and genetically interact in peripheral nerve. (A) GTP-RhoA assay to assess Gpr56 signaling in response to exogenous ligands. Addition of collagen III, a known activating ligand of Gpr56, to HEK 293T cells coexpressing Gpr56 result in elevation of GTP-RhoA levels over control (untransfected). Addition of prion protein (FT) does not induce Gpr56 signaling through RhoA. (B) Gpr56 Presto-Tango luciferase assay to assess Gpr56 signaling in HTLA cells. Addition of collagen III results in a significant elevation of luciferase compared with AcOH alone control (*, P < 0.05, unpaired Student’s t test). Addition of FT did not result in a significant elevation of luciferase relative to PBS controls (P = 0.0647, unpaired Student’s t test). (C) cAMP ELISA assay to assess cAMP signaling through Gpr56 and Gpr126 (positive control) after addition of FT. Addition of FT to HEK 293T cells expressing Gpr126 results in a significant elevation of cAMP (**, P < 0.01, unpaired Student’s t test) compared with PBS controls. Addition of FT to cells expressing Gpr56 does not induce cAMP signaling (P = 0.4575, unpaired Student’s t test). (A–C) Data are representative of three biologically independent experiments. Error bars represent SEM. (D) Co-IP to assess interactions between GPR56 and plectin in P5 WT SNs (n = 8 nerves from four animals per replicate; n = 3 replicates). Lane 1: plectin coimmunoprecipitates with GPR56 when the NTF of GPR56 fused to human IgG Fc (NTF-hFc) is used as a bait but not when pulling down with hFc alone (lane 2). (E) Analysis of plectin levels in P6 SNs. Plectin is significantly increased in Gpr56−/− nerves over sibling controls (**, P < 0.002, unpaired Student’s t test, n = 12 nerves from six animals; error bars represent SEM). (F) Graphic model: Gpr56 promotes function of developing and mature SCs. During early SC development, Gpr56 signals through Gα12/13 and RhoA to modulate the actin cytoskeleton for efficient radial sorting of axons. During SC maturation/homeostasis, Gpr56 functions upstream of SREBP1 to regulate myelin thickness and Remak SC biology, potentially through SRE signaling downstream of RhoA (gray dashed arrow). Concordantly, Gpr56 regulates mature myelin domain organization and myelin maintenance through direct interactions with the cytolinker protein plectin, which has been shown to physically interact with additional regulators of myelin domain organization (utrophin and β-dystroglycan). We therefore hypothesize that plectin serves as a docking center for cell signaling molecules that govern the partitioning of myelin into mature subdomains (Cajal bands and appositions) and helps generate these structures through its interactions with the cytoskeleton.

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