The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist

Abstract

Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Although the physiologic importance of aGPCRs was demonstrated in several studies, the majority of these receptors is still orphan with respect to their agonists and signal transduction. Recent studies reported that aGPCRs are activated through a tethered peptide agonist, coined the Stachel sequence. The Stachel sequence is the most C-terminal part of the highly conserved GPCR autoproteolysis-inducing domain. Here, we used cell culture-based assays to investigate 2 natural splice variants within the Stachel sequence of the orphan G s coupling aGPCR GPR114/ADGRG5. There is 1 variant constitutively active in cAMP assays (∼25-fold over empty vector) and sensitive to mechano-activation. The other variant has low basal activity in cAMP assays (6-fold over empty vector) and is insensitive to mechano-activation. In-depth mutagenesis studies of these functional differences revealed that the N-terminal half of the Stachel sequence confers the agonistic activity, whereas the C-terminal part orientates the agonistic core sequence to the transmembrane domain. Sequence comparison and functional testing suggest that the proposed mechanism of Stachel-mediated activation is relevant not only to GPR114 but to aGPCRs in general.-Wilde, C., Fischer, L., Lede, V., Kirchberger, J., Rothemund, S., Schöneberg, T., Liebscher, I. The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist. FASEB J. 30, 666-673 (2016). www.fasebj.org

Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5

“…stalkless). This result aligns with previous results suggesting that multiple components of adhesion GPCR stalks that emanate from TM1 seem to be required for full tethered agonism; a component at the stalk N-terminus, which 3-a-DOG might substitute for, and a second component proximal to TM1 (Stoveken et al, 2015;Wilde et al, 2016). …”

“…The N-terminal seven amino acids (TYFAVLM) of the GPR56 and GPR114 tethered-peptide-agonist stalks are identical and both receptors are activated by long, 18-20 residue GPR114 synthetic agonist stalk peptides, even though the GPR114 peptide sequence completely diverges from GPR56 sequence after the first seven residues ( Fig. Supplemental Figure 3) (Wilde et al, 2016). Because the GPR110 tethered peptide agonist sequence TSFSILM differs from the GPR56/114 sequence, the observed 3-a-DOG inhibition of GPR110 suggests that the compound may access the GPR110 orthosteric site, but does so in a manner that inhibits the receptor, rather than acting as a partial agonist.…”

“…The final version may differ from this version. (Liebscher et al, 2014;Stoveken et al, 2015;Wilde et al, 2016). In order to develop aGPCR modulatory compounds as tenable therapeutic leads that target aGPCRs in vivo, higher affinity must be achieved.…”

“…3A). GPR114 elevated cellular cAMP levels and was later found to couple directly to Gs in response to its tethered-peptide-agonist (Stoveken et al, 2016;Wilde et al, 2016).…”

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein–Coupled Receptor Antagonist

“…It was recently discovered that the extracellular peptide stalks emanating from the first transmembrane-spanning helix of multiple adhesion GPCRs are tethered-peptide agonists that activate aGPCR-mediated heterotrimeric G protein signaling (Liebscher et al, 2014;Demberg et al, 2015;Stoveken et al, 2015;Wilde et al, 2016). Tethered-agonist regulation is intimately linked to the ability of aGPCRs to execute a precise autocatalytic, self-cleavage event in which the P19 residue becomes the N terminus of the ∼17-26 amino acid tethered-agonist stalks (Lin et al, 2004;Arac et al, 2012;Liebscher et al, 2014;Stoveken et al, 2015).…”