Figure 1.

Generation of noncleavable mutant GPR56. A, schematic of GPR56, including the PLL, pentraxin/laminin/neurexin/sex-hormone-binding-globulin-Like domain (blue), cleavable GAIN domain (gray; tethered agonist, green), and 7-transmembrane domain. The GAIN domain can be cleaved between amino acids 382 and 383. B, noncleavable mutant GPR56^H381S has no cleavage site in the GAIN domain. C, comparison of the tethered agonist regions of mouse and human GPR56. The tethered agonist (TA, green) is conserved between two species. The mutant sites of GPR56^H381S and GPR56^L382A are highlighted in red. The arrow indicates the cleavage site. D, expression of WT GPR56 and mutant GPR56 (GPR56^H381S and GPR56^L382A). Scale bar = 5 μm. E, no significant difference was observed among their expression. F and G, Western blotting of whole-cell lysates of cells expressing WT and mutant constructs. The presence of full-length GPR56 and cleaved fractions was determined with the GPR56 N-terminal antibody. IB, immunoblot. H, basal activity of WT and GPR56^H381S constructs as measured by the SRE–luciferase reporter assay. Data are presented as mean ± S.D.; n = 3; ****, p < 0.0001; ns, not significant; two-tailed Student's t test.