Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Abstract

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα_12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα_q/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

Keywords: G protein-coupled receptor (GPCR); arrestin; ion channel; receptor; signal transduction.

Figure 1.

Schematic diagrams of full-length and ΔNT versions of R565W and L640R ADGRG1 mutant receptors. The illustrations depict the predicted transmembrane architecture and relative positions of mutations on the extracellular loops for the FL-R565W G1 mutant (A), the truncated ΔNT-R565W mutant (B), the FL-L640R mutant (C), and the ΔNT-L640R mutant (D).

Figure 2.

R565W and L640R mutations have differential effects on the surface expression of full-length versus ΔNT ADGRG1. A and C, representative Western blots showing surface and total expression of R565W and L640R mutant receptors compared with their wild-type counterparts. The lower blot in each panel represents total receptor expression, and the upper blot in each panel represents the amount of receptor pulled down by streptavidin beads (“Strep”) following biotinylation of surface-expressed proteins. B and D, quantified results of three independent Western blot experiments demonstrating that both full-length mutants exhibit markedly reduced surface and total expression, whereas ΔNT mutants do not, relative to their wild-type counterparts (one-way analysis of variance; **, p < 0.01; ***, p < 0.001 for the indicated comparisons; error bars represent S.E.). IB, immunoblotting; CT, C terminus; NS, not significant.

Figure 3.

The exposed stalk of ADGRG1 does not act as a pharmacological chaperone. A and B, to test the idea that the exposed stalk of ΔNT might act as a pharmacological chaperone to counteract surface trafficking deficits conferred by mutations to the G1 extracellular loops, a stalkless version of ΔNT-L640R (B; SL-L640R) was developed. A representative Western blot (C) and the quantified results of three independent experiments (D) demonstrate that deletion of the ΔNT-L640R stalk does not impair receptor surface expression (n = 3; error bars represent S.E.). Strep, streptavidin; IB, immunoblotting; CT, C terminus; NS, not significant.

Figure 4.

R565W and L640R mutations have differential effects on ADGRG1 signaling. A, FL- and ΔNT-R565W and -L640R mutants failed to elicit significant signaling to SRF luciferase compared with mock-transfected cells, whereas wild-type G1 and ΔNT elicited substantial signaling. B, ΔNT and ΔNT-R565W/L640R displayed signaling to NFAT luciferase comparable with their wild-type counterparts. All signaling data shown here are from at least five independent experiments (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus cells transfected with a mock vector; error bars represent S.E.). A representative Western blot (C) and quantified results from three independent experiments (D) demonstrate that both wild-type ΔNT and ΔNT-L640R robustly co-immunoprecipitate with HA-βArr2. IB, immunoblotting; IP, immunoprecipitation; CT, C terminus; luc, luciferase; NS, not significant.

Figure 5.

β-Arrestin2 overexpression dampens ADGRG1-mediated activation of SRF but not NFAT luciferase. A, overexpression of FLAG-tagged β-arrestin2 with full-length or ΔNT G1 resulted in significant reductions in receptor-mediated activation of SRF luciferase. B, overexpression of FLAG-β-arrestin2 full-length or ΔNT G1 had no significant effect upon ADGRG1-mediated signaling to NFAT luciferase. Results are from five independent experiments (*, p < 0.05 compared with the corresponding receptor condition without FLAG-β-arrestin2; error bars represent S.E.). luc, luciferase; NS, not significant.

Figure 6.

Mutation of a putative phosphorylation site (S690A) on the C terminus of ADGRG1 enhances surface expression and signaling by the ΔNT mutant but does not abolish binding to β-arrestin2. A representative Western blot (A) and quantified results from three independent experiments (B) demonstrate that there was no significant difference in co-immunoprecipitation with β-arrestin2 between wild-type G1-ΔNT and ΔNT-S690A. A representative Western blot (C) and quantified results from three independent experiments (D) reveal that the S690A mutation enhanced the surface expression of the ΔNT mutant but not the full-length mutant. The ΔNT-S690A mutant also displayed significantly higher levels of SRF (E) and NFAT luciferase (F) activation compared with the wild-type ΔNT receptor (*, p < 0.05; **, p < 0.01 for the indicated comparisons; error bars represent S.E.). Results shown are from at least four independent experiments. IB, immunoblotting; IP, immunoprecipitation; CT, C terminus; NS, not significant; luc, luciferase; a.u., arbitrary units.

Figure 7.

ADGRG1-mediated signaling to NFAT luciferase involves activation of calcium channels but not receptor coupling to Gα_q/11. A, treatment with the phospholipase Cβ inhibitor U73122 (50 μm; 8 h) had no effect on ΔNT-mediated activation of NFAT luciferase. B, treatment with the calcium channel inhibitor SKF96365 (SKF) (10 μm; 8 h) ablated activation of NFAT luciferase by both G1-ΔNT and ΔNT-L640R. Results shown are from at least four independent experiments (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001 for the indicated comparisons; error bars represent S.E.). C, schematic model depicting the putative signaling pathways by which G1 stimulates SRF or NFAT luciferase activity. The N-terminal fragment is shown interacting with the extracellular stalk and potentially the extracellular loops of the transmembrane C-terminal fragment to modulate receptor signaling activity. NS, not significant; luc, luciferase; veh, vehicle; GEF, guanine nucleotide exchange factor.