Nature research paper: Structural basis of tethered agonism of the adhesion GPCRs ADGRD1 and ADGRF1
were cloned into the pFastBac1 vector with a 6×His tag adding to their N termini. The genes of human Gβwere subcloned into a pFastBac Dual vector . All mutants used for structural and functional studies were generated by using site-directed mutagenesis PCR.The G protein-bound ADGRD1 and ADGRF1 complexes were obtained by co-expressing the receptor, miniGα and Gβin High Five insect cells . The cells were routinely tested for mycoplasma contamination.
The cells expressing the ADGRD1– or ADGRF1–G protein complexes were collected by centrifugation and suspended in a buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MgCland EDTA-free protease inhibitor cocktail tablets using dounce homogenization. The suspended membrane solution was supplemented with 25 mU mlapyrase and incubated at room temperature for 2 h. The membrane pellets were collected by centrifugation at 20,000for 30 min.
The formation of ADGRD1– and ADGRF1–G protein complexes was confirmed by negative staining electron microscopy and the sample quality was evaluated by a 200 kV Talos Arctica G2 electron microscope . For data acquisition, 3 μl of purified complex sample was applied to glow-discharged 300-mesh gold grids and followed by vitrification via plunge-freezing in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV with 1.5 s blot time and 0 blot force at 4 °C and 100% humidity.
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