Site-directed anti-peptide antibodies define the topography of the β-adrenergic receptor

Molecular cloning has revealed the primary structure of a number of G-protein-linked receptors. The organization and topography of these proteins predicted to have seven hydrophobic membrane-spanning domains, in contrast, have not been established. Antibodies were prepared against 11 peptides corresponding to each of the hydrophilic sequences of the hamster β₂-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and β₂-adrenergic receptor (M(r) 65,000) on blots of cell membranes and of purified receptor. All but three anti-peptide antisera also displayed immunoreactivity toward human placental and rat fat cell β₁-adrenergic receptors, reflecting the level of sequence identity that exists between the two subtypes. Chinese hamster ovary cells stably transfected with an expression vector harboring the cDNA encoding the hamster β₂-adrenergic receptor provided a cell type with 2 million receptors/cell, suitable for in situ localization of the sequences used as antigens. Indirect immunofluorescence of intact and permeabilized cells performed with these site-directed anti-peptide antibodies permitted the assignment of the general topography of each of the hydrophilic sequences of this G-protein-linked receptor. The results support the predictive value of hydropathy analysis for one class of membrane proteins with multiple transmembrane-spanning domains.