ADGRF1 and miniGαs/q were co-expressed in Expi293F cells. Cells were seeded at a density of 3.3 x 106 cells/mL in 900 mL of Expi293 expression media. To transfect cells, equal amounts of receptor (250 ug) and miniG (250 ug) DNA were resuspended in 50 mL opti-MEM, FectoPro (Polyplus) transfection reagent (0.5 mL) was resuspended in 50 mL opti-MEM, the DNA and FectoPro solutions were mixed to give a final 1:1 DNA/FectoPro ratio, and the mixture incubated at room temperature for 10 minutes before adding to cells. Approximately 24 hours later, filter-sterilised Valproic acid sodium salt (Sigma-Aldrich) was added to 5 mM, along with 10 mL of 45 % D-(+)- Glucose solution (Sigma-Aldrich). Cells were cultured for a further 24 hours before they were harvested by centrifugation at 4000 g for 15 minutes. The pellet was flash frozen and then stored at -80 o 294 C until purification.

Adhesion G Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to effect cell signalling. We report here that ADGRF1 can signal through all major G protein classes and identify the structural basis for a previously reported Gα_q preference by cryo-EM. Our structure shows that Gα_q preference in ADGRF1 may derive from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helix VII and helix VIII at the site of Gα recruitment. Mutational studies of the interface and of contact residues within the 7TM domain identify residues critical for signalling, and suggest that Gα_s signalling is more sensitive to mutation of TA or binding site residues than Gα_q. Our work advances the detailed molecular understanding of aGPCR TA activation, identifying features that potentially explain preferential signal modulation.