Complement factor 5a receptor chimeras reveal the importance of lipid-facing residues in transport competence.

Residues that mediate helix-helix interactions within the seven transmembranes (TM) of G protein-coupled receptors are important for receptor biogenesis and the receptor switch mechanism. By contrast, the residues directly contacting the lipid bilayer have only recently garnered attention as potential receptor dimerization interfaces. In the present study, we aimed to determine the contributions of these lipid-facing residues to receptor function and oligomerization by systemically generating chimeric complement factor 5a receptors in which the entire lipid-exposed surface of a single TM helix was exchanged with the cognate residues from the angiotensin type 1 receptor. Disulfide-trapping and bioluminescence resonance energy transfer (BRET) studies demonstrated robust homodimerization of both complement factor 5a receptor and angiotensin type 1 receptor, but no evidence for heterodimerization. Despite relatively conservative substitutions, the lipid-facing chimeras (TM1, TM2, TM4, TM5, TM6 or TM7) were retained in the endoplasmic reticulum/cis-Golgi network. With the exception of the TM7 chimera that did not bind ligand, the lipid-facing chimeras bound ligand with low affinity, but similar to wild-type complement factor 5a receptors trapped in the endoplasmic reticulum with brefeldin A. These results suggest that the chimeric receptors were properly folded; moreover, native complement factor 5a receptors are not fully competent to bind ligand when present in the endoplasmic reticulum. BRET oligomerization studies demonstrated energy transfer between the wild-type complement factor 5a receptor and the lipid-facing chimeras, suggesting that the lipid-facing residues within a single TM segment are not essential for oligomerization. These studies highlight the importance of the lipid-facing residues in the complement factor 5a receptor for transport competence.