Is helix VIII of G protein‐coupled receptors (GPCRs) a lipid‐activated signalling sensor?

The inactive rhodopsin GPCR structure confirmed an additional helix (H8) within the proximal C‐terminus, positioned parallel to the membrane. We have identified that H8 of the angiotensin II receptor interacts with membranes with high affinity and identify residues within TM7 and H8 that may assist positioning H8 towards the membrane. Two approaches were undertaken to investigate this interplay by introducing: Cysteine residues to form a disulfide bond between TM7 and H8. An unnatural palmitoylation site to anchor H8 to the membrane. Results show H8 may not detether from the membrane but undergoes a subtle conformational change. An alanine scan was conducted along H8 to identify residues important for membrane tethering and receptor function. Some mutations displayed reduced (L305A to K308A, L316A, P321A and P322A), unchanged (F304A, F309A to K311A and Y319A), and enhanced (Y312A to L314A) receptor expression. In order to identify the influence of each residue on folding, cell‐surface trafficking, receptor ligand affinity and specific membrane‐lipid targeting, mutant receptors are subjected to ligand binding, internalisation and signalling assays, in conjunction with surface plasmon resonance assays of H8 peptide using liposomal surfaces with diverse lipid compositions. Preliminary results show that H8 and its positioning with respect to membranes is crucial for GPCR function.