G protein Coupled Receptors (GPCRs) Reconstituted on Recombinant Proteoliposomes using Baculovirus-Liposome Membrane Fusion

摘要

Transmembrane proteins are important in biological functions. Proteoliposomes, which contain reconstituted membrane proteins, have been considered to be useful for their investigation. Especially, cell-sized giant liposomes, or giant unilamellar vesicles, are often used for such purposes. Previously, we established a novel method of proteoliposome preparation using membrane fusion between recombinant baculovirus and liposomes. Here we demonstrated preparation of recombinant proteoliposomes containing a G protein coupled receptor (GPCR) using this method. Human corticotropin releasing hormone receptor 1 (CRHR1), which is a seven-span transmembrane protein, was expressed using a baculovirus/insect cell recombinant system, and it was verified that the proteins were localized on both the insect cell membranes and baculovirus budded virus envelopes. The budded viruses were fused with GUVs containing DOPC/DOPS at pH ～4.5. The resulting proteo-GUVs were visualized using phycoerythrin-conjugated anti-CRHR antibodies. The CRHR1 recombinant proteoliposomes also reacted with anti-ligand antibodies in the presence of its ligand (corticotropin releasing factor). These results suggest that GPCRs can be reconstituted on proteoliposomes with an intact (native) function and structure using the baculovirus-liposome membrane fusion method.