GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist

Abstract

Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.

Keywords: 3-α-DOG; ADGRG1/GPR56; G protein–coupled receptor (GPCR); GPCR autoproteolysis-inducing (GAIN) domain; TG2; cell signaling; collagen; development; shedding; tethered agonist.

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.

Figure 2.

GAIN domain cleavage is required for GPR56 activation. A, cells transfected with WT GPR56 and GPR56^H381S were treated with 3-α-DOG (5 μm), P19 (20 μm), or collagen III (Col III, 50 nm) in an SRE–luciferase reporter assay. Data are normalized to the Renilla luciferase control and expressed as -fold over the signal obtained from cells transfected with SRE–luciferase only. Data are presented as mean ± S.D.; n = 3; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; two-tailed Student's t test. B and C, RhoA activation by treatment with 3-α-DOG (5 μm), P19 (20 μm), or collagen III (50 nm) on WT GPR56-expressing HEK293T cells. Data are presented as mean ± S.D.; n = 3; *, p < 0.05; **, p < 0.01; two-tailed Student's t test.

Figure 3.

TG2 requires the presence of the ECM protein laminin to activate GPR56. A, TG2 promotes oligodendrocyte precursor cell proliferation in an GPR56- and laminin (Lam)-dependent manner. B, cells transfected with WT GPR56 and GPR56^H381S were treated with TG2 (200 nm), laminin (2 μg/ml), or TG2 plus laminin in an SRE–luciferase reporter assay. Data are normalized to Renilla luciferase control and expressed as -fold over the signal obtained from cells transfected with SRE–luciferase only. Data are presented as mean ± S.D.; n = 3; **, p < 0.01; ns, not significant; two-tailed Student's t test. C and D, RhoA activation by treatment with TG2 (200 nm) or TG2 plus laminin (2 μg/ml) of WT GPR56-expressing HEK293T cells. Data are presented as mean ± S.D.; n = 3; **, p < 0.01; two-tailed Student's t test. Fib, fibronectin.

Figure 4.

Dissociation and shedding of GPR56 NTF. A and B, HEK293T Cells transfected with WT GPR56 were treated with TG2 (200 nm), TG2 plus laminin (2 μg/ml), 3-α-DOG (5 μm), P19 (20 μm), or collagen III (50 nm). Western blotting reveals the NTF of GPR56 in the supernatant. IB, immunoblot. C and D, quantification of GPR56 NTF in the supernatant. Data are presented as mean ± S.D.; n = 3; *, p < 0.05; ***, p < 0.001; ns, not significant; two-tailed Student's t test.

Figure 5.

The activation mechanism of 3-α-DOG on GPR56. A, AmpC β-lactamase inhibition response curve with different concentrations of 3-α-DOG. B, critical aggregation concentration curve for 3-α-DOG by dynamic light scattering assay. C, 3-α-DOG inhibits P19 responses. SRE–luciferase reporter assay responses were normalized to the maximal P19 response in the absence of 3-α-DOG. Data are presented as mean ± S.D.; n = 3; ****, p < 0.0001; two-tailed Student's t test. D, summary of 3-α-DOG (orange triangle) activity on various truncated GPR56 7TM receptor (42). The seven-amino-acid Stachel is depicted as seven solid green circles (each represents one amino acid).

Figure 6.

Schematic of the GPR56 cleavage-dependent signaling pathway. A, collagen III binds to GPR56 NTF, causing conformational changes, including NTF shedding. Exposure of the Stachel tethered agonist results in 7TM domain activation. B, TG2 binds to GPR56 but fails to activate the receptor in the absence of the ECM protein laminin. C, combined binding of TG2 and laminin to GPR56 induces dissociation of GPR56 NTF from its CTF, allowing Stachel to initiate downstream signaling. D, P19 possesses the seven amino acids of the tethered agonist and activates downstream signaling. E, 3-α-DOG binds to part of the orthosteric site of the receptor and activates GPR56 when the other pocket of the ligand binding site is engaged by part of Stachel. Stachel of the noncleavable receptor GPR56^H381S is buried by the NTF and therefore cannot be activated by 3-α-DOG.