Site-directed anti-peptide antibodies define the topography of the beta-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 beta 2-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and beta 2-adrenergic receptor (Mr 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 beta 1-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 beta 2-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.
Wang
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H
Department of Pharmacology, State University of New York, Stony Brook 11794-8651.
Lipfert
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L
Malbon
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CC
Bahouth
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D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005110:Extracellular Space / Q000032:analysis; D005260:Female; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D015151:Immunoblotting; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010053:Ovary; D010455:Peptides / Q000276:immunology*; D011487:Protein Conformation; D011943:Receptors, Adrenergic, beta / Q000276:immunology / Q000302:isolation & purification*; D013194:Staining and Labeling
D007106:Immune Sera; D015335:Molecular Probes; D010455:Peptides; D011943:Receptors, Adrenergic, beta