Extended Data Fig. 5: Urea-mediated ADGRL3 N-terminal Fragment dissociation.
From: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3
For the membrane urea treatment experiments presented in this figure, a FLAG- (N-terminal) and His8- (C-terminal) tagged ADGRL3 construct that was truncated N-terminally to the HormR domain was used21 (See Fig. 1 for Adgrl3 architecture). Insect cell membranes (High-Five) with expressed ADGRL3 were mock treated or extracted with urea. The presence of the ADGRL3 NTF and CTF in the membrane (Mem) and extract (Soluble, Sol) fractions was determined by immunoblotting with an anti-FLAG antibody to detect the NTF and an anti-penta-His antibody to detect the CTF. The NTF (apparent MW ~50 kDa) was partially solubilized with the urea, whereas the CTF (apparent MW ~27 kDa) was not. The penta-His blot panels are from one contiguous blot, but broken to avoid oversaturation of the ~70 kDa band (unprocessed receptor) and to show a higher exposure of low MW panel (~27 kDa CTF). Data from one representative experiment that was repeated three times.
