Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1)

Abstract

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and β-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.

Keywords: G protein-coupled receptor (GPCR); arrestin; proteolysis; receptor structure-function; signal transduction; ubiquitylation (ubiquitination).

FIGURE 1.

Tethered Cryptic Agonist Model of Adhesion GPCR Activation. A, according to this model, the unstimulated receptor is inactive due to the masking of an agonistic region of the stalk by the NTF. B, following ligand binding to the NTF, the NTF is released from the seven-transmembrane CTF to unveil a new N-terminal stalk, which then stimulates G protein-dependent signaling activity.

FIGURE 2.

GAIN domain cleavage is not necessary for G1 activity. A, schematic of T383A point mutation in G1. B, G1-T383A is expressed on HEK cell surface, albeit at a reduced level compared with the wild-type receptor. Molecular weight markers (in kDa) are shown on the left side of the blots. C, Western blots of G1 and G1-T383A reveal a ∼75 kDa band for G1-T383A that is both N-terminally and C-terminally reactive, suggesting non-cleavage of the mutant receptor. Equal amounts of protein (10–20 μg) were loaded in each lane for the blots shown in panels B and C, and these experiments were performed 3–4 times each. D and E, G1 and G1-T383A produce comparable activity in the AP-TGFα shedding and SRF-luciferase assays. Results for TGFα and SRF-luc are from 3–6 independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus cells transfected with empty vector, denoted by EV).

FIGURE 3.

Generation of G1 and B1 SL receptors. A and C, schematic of G1-SL and B1-SL alongside their ΔNT counterparts. B and D, SL mutants exhibit comparable surface expression in HEK cells to their ΔNT counterparts. Molecular weight markers (in kDa) are shown on the left side of the blots. For G1, prominent C-terminally reactive bands between ∼40–45 kDa correspond to monomeric 7TM regions. Lower molecular weight bands at ∼25 kDa (for full-length G1) and ∼20 kDa (for G1-ΔNT or -SL) likely represent further cleaved forms of the proteins and/or differential conformations. Equal amounts of protein (10–20 μg) were loaded in each lane for the blots shown here, and the data shown in this figure are representative of 3–4 experiments for each pair of mutants.

FIGURE 4.

G1-SL exhibits differential levels of signaling activity in distinct assays whereas B1-SL is consistently active. G1-SL exhibited significant signaling activity in the TGFα-shedding (A) and NFAT luciferase (C) assays but was found to not be significantly active in the SRF-luciferase assay (B). However, B1-SL was significantly active at comparable levels to B1ΔNT in all three assays (D: TGFα-shedding, E: SRF-luc, F: NFAT-luc) demonstrating the dispensability of the B1 post-cleavage stalk. All experiments performed in HEK cells. TGFα, SRF-luc, and NFAT-luc results are from 4–6 independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus cells transfected with empty vector, denoted by EV).

FIGURE 5.

Inhibitors of Gα_12/13 and Gβ/γ block ΔNT and -SL signaling activity. The RGS domain of p115RhoGEF, a Gα_12/13 inhibitor, as well as the Gβ/γ inhibitor gallein, significantly block ΔNT and -SL signaling to NFAT for both receptors (5A-B: G1, 5C-D: B1). Results are from 3–6 independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01 versus G1/B1).

FIGURE 6.

G1-SL and B1-SL couple to G proteins. Western blots of co-immunoprecipitation experiments in HEK cells demonstrating that ΔNT and -SL receptors (6A: G1, 6B: B1) robustly associate with Gα₁₃ whereas the full-length versions of the receptors do not. Equal amounts of protein (10–20 μg) were loaded in each lane for the blots shown here, and the results shown are from 3–4 independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01 versus G1/B1).

FIGURE 7.

G1-SL and B1-SL bind robustly to βArrestin2. Western blots of co-immunoprecipitation experiments in HEK cells with HA or FLAG-tagged β-arrestin2 revealed that ΔNT and -SL receptors bound to β-arrestin2 significantly more than full-length receptors. Molecular weight markers (in kDa) are shown on the left side of the blots. Equal amounts of protein (10–20 μg) were loaded in each lane for the blots shown here, and the results shown are from three independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus G1/B1).

FIGURE 8.

G1-SL and B1-SL are heavily ubiquitinated. Western blots of co-immunoprecipitation experiments with HA-ubiquitin demonstrated that ΔNT and -SL receptors were significantly more ubiquitinated than full-length receptors. Molecular weight markers (in kDa) are shown on the left side of the blots. Equal amounts of protein (10–20 μg) were loaded in each lane for the blots shown here, and the results are from three independent experiments (± S.E. shown, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus G1/B1).

FIGURE 9.

Allosteric Antagonist Model of aGPCR Activation. A, in this model, the NTF behaves as an allosteric antagonist in two ways: (i) masking the stalk region and (ii) directly antagonizing the constitutive stalk-independent activity possessed by the 7TM region. B, conformational change of the NTF induced by ligand binding is sufficient to allow for enhanced stalk-dependent activity. C, ligand binding can also result in either NTF dissociation or a conformational change that relieves the inhibitory constraint of the NTF upon the 7TM region, such that both stalk-dependent and stalk-independent pathways are activated.