Collagen VI Is a Gi-Biased Ligand of the Adhesion GPCR GPR126/ADGRG6 - PubMed (original) (raw)
Collagen VI Is a Gi-Biased Ligand of the Adhesion GPCR GPR126/ADGRG6
Caroline Wilde et al. Cells. 2023.
Abstract
GPR126/ADGRG6, a member of the adhesion G-protein-coupled receptor family, balances cell differentiation and proliferation through fine-tuning of intracellular cAMP levels, which is achieved through coupling to Gs and Gi proteins. While GPR126-mediated cAMP increase has been proven to be essential for differentiation of Schwann cells, adipocytes and osteoblasts, Gi-signaling of the receptor was found to propagate breast cancer cell proliferation. Extracellular ligands or mechanical forces can modulate GPR126 activity but require an intact encrypted agonist sequence, coined the Stachel. Even though coupling to Gi can be seen for constitutively active truncated receptor versions of GPR126 as well as with a peptide agonist derived from the Stachel sequence, all known N-terminal modulators have so far only been shown to modulate Gs coupling. Here, we identified collagen VI as the first extracellular matrix ligand of GPR126 that induces Gi signaling at the receptor, which shows that N-terminal binding partners can mediate selective G protein signaling cascades that are masked by fully active truncated receptor variants.
Keywords: ADGRG6; GPR126; adhesion GPCR; biased signaling; collagen VI; extracellular matrix ligand.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Figure 1
Overview of the GPR126/ADGRG6 and ways to activate Gs and Gi signaling pathways through it. (A) The N terminus of GPR126 contains the CUB (pink eclipse), a pentraxin domain (Pent, purple square) and the Sperm protein, Enterokinase and Agrin (SEA) domain (blue polygon), including the furin site (dotted line). The highly conserved GPCR autoproteolysis-inducing (GAIN) domain (turquois rectangle) contains the GPCR proteolysis site (GPS) at which the receptor is cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF). The CTF harbors the Stachel sequence (S, green rectangle) at its extracellular portion. (B) GPR126 can signal through Gs protein by stimulation with either _Stachel_-sequence-derived peptides (pGPR126), small molecule apomorphine, anti-HA antibody (anti-HA ab) targeting an N-terminal inserted HA tag or through its ligands collagen IV, prion protein (PrPc) or laminin 211 in combination with mechanical forces. The small molecules progesterone and 17OHP, but also pGPR126, activate GPR126 via the Gi signaling pathway. Figure was created with biorender.
Figure 2
Collagen VI binds to the NTF of GPR126. (A) Summary of mass spectrometry results. Total number of peptide sequences specific for binding to GPR126-NTF. (B) NTF protein parts used for co-IP. (C) co-IP of GPR126 NTF with wild-type sciatic nerves, probed with anti-collagen VI antibody, demonstrates binding of collagen VI with GPR126 NTF-446-807 fragment. Detected bands for the fusion proteins are slightly bigger than theoretically estimated, indicating potential glycosylation. IB, immunoblotting.
Figure 3
Collagen VI is a new ligand of GPR126 with activating properties in the Gi-signaling pathway. (A) Concentration–response curves upon stimulation of wild-type GPR126 or vector control with collagen VI are shown as % of unstimulated vector control cAMP level (vc: cAMP level: 1.46 ± 0.3 nM = 100%). cAMP accumulation is displayed in the presence or absence of 2.5 µM forskolin co-stimulation in COS-7 cells as means ± SEM of three independent experiments each performed in triplicates. (B,C) Pretreatment with PTX abolishes reduction in cAMP through incubation with collagen VI (2 µg/mL) in cells stimulated (B) with or (C) without Fsk (2 µM). Of note, stimulation with the agonistic peptide pGPR126 (1 mM) shows significant increase of cAMP in assay conditions without Fsk co-stimulation (C). Data are shown as fold over unstimulated vector control (vc: cAMP level: 4.76 ± 1.45 nM) as means ± SEM of four independent experiments each performed in triplicates. Statistics were performed using paired _t_-test; * p < 0.05, ** p < 0.01. (D) COS-7 cells were transfected with wild-type, T841A Stachel mutant GPR126 or vector control. Accumulation of IP1 with Gqi chimera was measured after treatment of the transfected cells with or without collagen VI (2 µg/mL). Data are shown as fold over unstimulated vector control (vc: IP1 level: 6.37 ± 3.5 nM) as means ± SEM of three independent experiments each performed in triplicates. Statistics were performed using paired _t_-test; * p < 0.05.
Figure 4
Collagen-VI-mediated Gi activation is not amplified through additional shaking forces. (A) Accumulation of cAMP or (B) IP1 with Gqi chimera was measured after treatment of the transfected cells without mechanical forces (basal) or detachment of the transfected cells followed by mechano-activation at 100 rpm or through the combined application of mechanical forces and collagen VI (2 µg/mL Col VI). Vector control (vc) served as negative control (vc; cAMP level: 3.26 ± 0.2 nM; vc; IP1 level Gqi: 8.48 ± 3.74 nM). Data are given as x-fold over vector control as means ± SEM of at least three independent experiments each performed in triplicates. Statistics were performed using paired _t_-test; * p < 0.05, ** p < 0.01, *** p < 0.001.
References
- Liebscher I., Schön J., Petersen S.C., Fischer L., Auerbach N., Demberg L.M., Mogha A., Cöster M., Simon K.-U., Rothemund S., et al. A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133. Cell Rep. 2014;9:2018–2026. doi: 10.1016/j.celrep.2014.11.036. -DOI -PMC -PubMed
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