Antibodies to a Human α₂-C10 Adrenergic Receptor Fusion Protein Confirm the Cytoplasmic Orientation of the V-VI Loop

DNA encoding the hydrophillic region between transmembrane domains V and VI of the human platelet α₂-adrenergic receptor (α₂-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST). Expression of the recombinant plasmid in E. coli resulted in the production of a GST/α₂-C10 fusion protein which was purified by preparative SDS-PAGE. Chickens innoculated with the fusion protein produced antibodies that were present in their eggs. In cells expressing the α₂-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence. For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent. This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.