A Fusion Protein Platform for Analyzing Tethered Agonism in the Adhesion Family of G Protein-Coupled Receptors

Adhesion G Protein-Coupled Receptors (aGPCRs) are a distinct family of transmembrane proteins that carry out numerous cellular functions in multicellular organisms. During maturation, aGPCRs undergo autoproteolysis to generate non-covalently associated N-terminal and C-terminal fragments (NTF and CTF, respectively). Signaling is induced when a tethered agonist at the N-terminus of the CTF is released from or unmasked by the NTF to access its binding site within the seven transmembrane domain of the CTF. Here, we show that the tethered sequence does not require a free N-terminus to signal and exploit this finding to create a fusion protein platform for interrogation of structure-function relationships in aGPCRs. We analyze tethered agonism in CD97, a protein implicated in immune cell function and cancer cell invasiveness, and demonstrate the platform’s generality by creating constitutively active forms of several other aGPCRs. This platform should find wide applicability in analyzing signals transmitted by tethered agonists in aGPCRs. ### Competing Interest Statement S.C.B. is on the scientific advisory board for and receives funding from Erasca, Inc., is an advisor to MPM Capital, and is a consultant for Scorpion Therapeutics, Odyssey Therapeutics, Droia Ventures, and Ayala Pharmaceuticals for unrelated projects. A.C.K. is a co-founder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute.