Abstract Background: ADGRL4 (also known as ELTD1) is an orphan adhesion G protein-coupled receptor (aGPCR) upregulated in multiple aggressive malignancies where it promotes angiogenesis, proliferation and epithelial-mesenchymal transition. Its molecular activation mechanism however was previously unknown. We demonstrated that ADGRL4 couples weakly to the heterotrimeric G protein Gq and determined the first active-state cryo-EM structure of ADGRL4. We then combined the structural data with systematic mutagenesis and G protein recruitment assays to define the molecular determinants of tethered agonist (TA) mediated activation. Methods: A split NanoLuc-proximity assay using engineered ADGRL4 and mini-G protein constructs was used to quantitate Gq coupling. We purified the active-state ADGRL4-mini-Gq-βγ complex and determined its structure by cryo-EM. Guided by the structure, we performed alanine-scanning mutagenesis on TA residues contacting the orthosteric binding pocket and selected residues within the receptor core, assessing effects on Gq recruitment in HEK293T cells. Results: Cryo-EM analysis yielded the first high-resolution structure of active-state ADGRL4 to 3.1 Å resolution. The TA region forms a short α-helix that occupies the orthosteric site and promotes outward displacement of transmembrane helix 6 (TM6). Alanine substitution of seven out of eight TA residues that interact with the orthosteric pocket significant impaired Gq recruitment. Mutation of six residues (H408A, F409A, I411A, L412A, M413A, S414A) reduced coupling by 50%; M413A abolished coupling completely (p0.0001) and F409A reduced coupling by 88% (p0.0001). Previous aGPCR structures have proposed three conserved core activation motifs: the upper quaternary core motif (UQC), the hydrophobic P/F/W/LφφG motif, and the H(N)L(M)Y motif. Structure-guided mutagenesis of corresponding positions in ADGRL4 showed that mutation of any UQC residue to alanine (F505A, M508A, W631A) abolished Gq recruitment. In the P/F/W/LφφG motif, L627A and G628 eliminated activity, whilst F625A and L626A caused partial impairment. In the H(N)L(M)Y motif, L515A and Y516A abolished coupling, whilst H514A produced partial impairment. Together, these findings clarify ADGRL4’s activation mechanism: exposure of the TA permits engagement with the orthosteric pocket, which in turns facilitates TM6 displacement and formation of a cytoplasmic cleft for Gq engagement, with all activation motifs functioning as essential structural elements of this process. Conclusions: These findings define how ADGRL4’s TA and conserved 7TM activation motifs cooperatively stabilise its active conformation and engage Gq. These findings establish a structural framework for developing ADGRL4-selective antagonists (nanobodies, synthetic binders or small molecules) with potential therapeutic utility in ADGRL4-associated malignancies. Citation Format: David M. Favara, Qingchao Chen, Anastasiia Gusach, Aurora Diamante, Jayesh C. Patel, Patrcia C. Edwards, Christopher Tate. The cryo-EM structure of ADGRL4 provides functional insights into its mechanism of activation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2426.
Favara et al. (Fri,) studied this question.