The Adhesion GPCR GPR114/ADGRG5 is Activated by its Tethered-Peptide-Agonist Following Receptor Fragment Dissociation

Abstract

<b>Abstract ID 17396</b> <b>Poster Board 478</b> The second largest group of G protein coupled receptors (GPCRs) is the class B2 group or adhesion GPCRs (AGPCRs). AGPCRs are distinguished by variable extracellular regions that contain a membrane-proximal GPCR autoproteolysis-inducing (GAIN) domain that self cleaves to generate two receptor fragments, the N-terminal fragment (NTF) and the C-terminal fragment (CTF), or seven transmembrane domain (7TM). The fragments remain non-covalently bound after self-cleavage. We hypothesize that binding of the NTF to its ligand(s) that are presented by a neighboring cell or the extracellular matrix, anchors the NTF, while the AGPCR-expressing cell moves to generate force that dissociates the NTF/CTF to expose the AGPCR tethered-peptide-agonist (TA). The decrypted TA rapidly binds to its orthosteric site within the CTF to stabilize the AGPCR active state. The orphan AGPCR GPR114 was purported to be a non-cleaved, yet still capable of TA-dependent activation. This prompted confounding models of AGPCR activation, such as the TA was proposed to reside simultaneously within the GAIN domain and the CTF orthosteric site. This seems improbable as GPR114 has an identical cleavage site and TA as its closest homolog, the efficiently-cleaved AGPCR, GPR56. Here we used immunoblotting of dually epitope-tagged and untagged GPR114 to show that it is a cleaved AGPCR. Furthermore, FLAG-tagged GPR114 NTF was isolated from the culture medium of suspension cells expressing the holoreceptor, demonstrating its ability to be spontaneously shed from the cell surface. Urea treatment of AGPCR membrane homogenates is an established proxy means to induce AGPCR NTF dissociation and invoke tethered agonism. The wild type receptor, but not cleavage-defective GPR114-H225S, was activated by urea treatment to induce strong Gs activation. GPR114-H225S was activated by the AGPCR Group VI selective small molecule agonist 3-a-DOG or a synthetic peptidomimetic of its tethered agonist. Finally, engineered protease activated receptor (PAR1) / AGPCR fusion proteins of GPR114 and GPR56 were acutely stimulated with thrombin protease and shown to activate Gs and G13 respectively. Together, these results show that the GPR114 activation process typifies that of other AGPCRs and requires NTF/CTF dissociation to permit the tethered agonist to bind its CTF orthosteric site. Additional evidence demonstrating endogenous GPR114 self-cleavage mediated activation in primary cells will be presented.