Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

Abstract

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

Figure 1

Conserved residues in the extracellular loop 2 (ECL2) of aGPCRs. (a) Multiple alignments of the ECL2 of all 33 members of the aGPCR family. Predicted amino acid sequences of the ECL2 for each aGPCR were derived from GPCRdb. The alignment was conducted in SnapGene software (from Insightful Science; available at www.snapgene.com) using the Clustal Omega algorithm. (b) Snakeplot of human ADGRG2, exported from www.GPCRdb.org, showing the colored CWI motif in the ECL2. The sequences of the N-terminal fragment and C-terminus are not shown. The dashed line shows the disulfide bond between the cysteines of TM3 and ECL2.

Figure 2

Expression of ADGRG2-P622 is regulated by the residues in the CWI motif in the ECL2. HEK cells were transfected with the same dose (50 ng for a–c; 1 µg for d) of plasmids expressing P622-CWI or mutants. Cell surface expression of receptors was determined by (a) ELISA and (b) immunofluorescence imaging using an antibody against the N-terminal HA-tag in non-permeabilized conditions. Nuclear counterstaining with DAPI (scale bars: 20 µm). (c) ELISA was used to measure the total expression by using an antibody against the C-terminal V5 tag in permeabilized conditions. (d) Representative blots show C-terminal V5-tagged P622-CWI and mutant receptors in total cell lysates. Densitometry data were used to normalize V5 expression to β-actin for each plasmid and are shown as a percentage of P622-CWI. Uncropped blots are provided in Supplementary Fig. 5. Data are mean ± S.E.M from a representative experiment out of 4 (for a and c, performed in quadruplicate) independent experiments. Images (b and d) are representative of 3 independent experiments. One-Way ANOVA with Dunnett’s multiple comparison test was used for statistical analyses. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

Degradation of ADGRG2-P622 is regulated by the residues in the CWI motif in the ECL2. HEK cells were transfected with the same dose of plasmids (1 µg) expressing P622-CWI or mutants. Cells were then incubated with cycloheximide (100 µg/ml) for up to 8 h and total cell lysates were run on SDS-PAGE, transferred to PVDF membranes, and probed with V5 and β-actin antibodies. Differential exposure time in the iBright Imaging system was used to make the mutants visible to facilitate the band density quantification. Representative blots show a reduction of mutant receptor levels over time. Density values of V5 were normalized to that of β-actin for each plasmid at NT (0 h). Densitometry analyses are shown on the right. Data are mean ± S.E.M from 4 independent experiments. Uncropped blots are provided in Supplementary Fig. 5. Two-Way ANOVA with Dunnett’s multiple test was used to compare protein levels at each time point to that of basal expression (NT) of respective receptors. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Proteasomal but not endosomal or lysosomal pathways control degradation of ECL2 mutants. (a) HEK cells were transfected with the same dose of plasmids (1 µg) expressing P622-CWA, P622-CAI, and P622-CAA. Cells were then pre-incubated with chloroquine (CQ; lysosomal acidification inhibitor, 25 µM), MG-132 (MG; proteasomal inhibitor, 1 µM), and bafilomycin A1 (Baf; inhibitor of v-ATPase, endosomal acidification, and late-stage vesicle maturation, 10 nM) overnight, followed by an 8-h incubation with cycloheximide (100 µg/ml). Total cell lysates were run on SDS-PAGE, transferred to PVDF membranes, and probed with V5 and β-actin antibodies. Representative blots from 3 independent experiments are shown. Uncropped blots are provided in Supplementary Fig. 5.

Figure 5

ECL2 plays a major role in ADGRG2 activation by synthetic Stachel peptide. (a) HEK cells were transfected with one dose of P622-CWI (50 ng) and different doses of mutant P622 plasmids. Cell surface expression of receptors was determined by ELISA using an antibody against the N-terminal HA-tag in non-permeabilized conditions. X denotes the amount of mutant plasmids that resulted in comparable surface expression as 50 ng P622-CWI plasmid. Data are mean ± S.E.M from a representative experiment out of 3 independent experiments performed in quadruplicate. (b) Cell surface expression of receptors, after transfection with the adjusted doses of plasmids, was determined by immunofluorescence imaging using an antibody against the N-terminal HA-tag in non-permeabilized conditions. Nuclear counterstaining with DAPI. Representative images from 3 independent experiments are shown (scale bars: 20 µm). (c, d) Cells were transfected with adjusted doses of either P622-CWI or mutant plasmids along with either CRE-Luc (c) or SRE-Luc (d) plasmids. After an overnight of serum starvation, cells were activated with increasing concentrations of P-15 for 5 h. Luciferase induction was measured in a luminescence-based assay. Relative light units (RLU) recorded in a luminometer are representative from 3 independent experiments performed in triplicate and are presented as mean ± S.E.M.

Figure 6

ECL2 is essential for the constitutive activity of NTF-truncated ADGRG2. (a) HEK cells were transfected with one dose of ∆NTF-CWI (50 ng) and different doses of mutant ∆NTF plasmids. Cell surface expression of receptors was determined by ELISA using an antibody against the N-terminal HA-tag in non-permeabilized conditions. X denotes the amount of mutant plasmids that resulted in comparable surface expression as 50 ng ∆NTF-CWI plasmid. Data are mean ± S.E.M from a representative experiment out of 3 independent experiments performed in triplicate. (b) Cell surface expression of receptors, after transfection with the adjusted doses of plasmids, was determined by immunofluorescence imaging using an antibody against the N-terminal HA-tag in non-permeabilized conditions. Nuclear counterstaining with DAPI. Representative images from 3 independent experiments are shown (scale bars: 20 µm). (c) Cells were transfected with adjusted doses of either ∆NTF-CWI or mutant plasmids and pcDNA3.1 as control. Basal cAMP production was measured after overnight incubation with 0.5 mM IBMX in starvation media in an HTRF assay. cAMP production in nM is presented as mean ± S.E.M from a representative experiment out of 3 independent experiments performed in duplicate. Data were compared with ∆NTF-CWI with one-Way ANOVA with Dunnett’s test. ****P < 0.0001. (d) Cells were transfected with adjusted doses of either ∆NTF-CWI or mutant plasmids along with CRE-Luc plasmid. After an overnight of serum starvation, cells were activated with either vehicle (DMSO) or 100 µM of P-15 for 5 h. Luciferase induction was measured in a luminescence-based assay. Relative light units (RLU) recorded in a luminometer are mean ± S.E.M from a representative experiment from 3 independent experiments performed in triplicate. Data were compared between vehicle and P-15 for each plasmid with two-Way ANOVA with Sidak’s multiple comparison test. ****P < 0.0001; ns: not significant.

Figure 7

ECL2-mutated ADGRG2-P622 receptors are not activated by P-15. (a–d) Cells were transfected with adjusted doses of P622-CWI and mutant plasmids and were transduced with a cAMP biosensor overnight. After a 20-s basal recording of fluorescence (Ex: 488 nm; Em: 525 nm), cells were activated with increasing concentrations of P-15 (arrowheads), and fluorescence was recorded for another 280 s. Relative fluorescence unit (RFU) data were analyzed in GraphPad Prism and are presented as change in RFU divided by the initial RFU (ΔF/F0). (e) Cells transfected with adjusted doses of plasmids and biosensor were stimulated with 1 µM forskolin (FSK). Data are mean ± S.E.M and are representative of 3 independent experiments performed in triplicate. (f) Basal cAMP production was measured after overnight incubation with 0.5 mM IBMX in starvation media in an HTRF assay. cAMP production in nM is presented as mean ± S.E.M from a representative experiment out of 3 independent experiments performed in triplicate. Data were compared with P622-CWI with one-Way ANOVA with Dunnett’s test. ns: not significant. (g) Cells were stimulated with increasing concentrations of P-15 for 2 h and cAMP production in nM was measured in an HTRF assay. Data are mean ± S.E.M from a representative experiment from three independent experiments performed in duplicate.

Figure 8

Whole-cell impedance assay shows a lack of activation of the ECL2-mutated ADGRG2 receptor by the Stachel peptide. Cells were transfected with adjusted doses of WT and mutant plasmids and were then seeded in plates that have electrodes at the bottom of each well to apply various frequencies of electric voltages. Serum-starved cells, at monolayer confluency, were kept in Maestro Z device until the recorded impedance (Ω) reached a steady state. Cells were stimulated with either vehicle (DMSO) or 100 µM P-15 and impedance was recorded for up to 2 h. Data are corrected to that of the vehicle for each plasmid and are mean ± S.E.M from a representative experiment out of 3 independent experiments conducted in quadruplicate.