Antipeptide Antibodies to the Beta 2-adrenergic Receptor Confirm the Extracellular Orientation of the Amino-terminus and the Putative First Extracellular Loop

Overview

Journal J Membr Biol

Date 1989 Oct 1

PMID 2559200

Citations 2

Authors

M A Theveniau

J R Raymond

G N Rougon

Affiliations

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Abstract

We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung beta 2-adrenergic receptor (amino acids 16-31 and 174-189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified beta 2-adrenergic receptor in immunoblot conditions when used at a dilution of 1:500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as 125I-cyanopindolol labeled beta 2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the 125I-cyanopindolol binding domain of the beta 2-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified beta 2-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native beta 2-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing beta 2-adrenergic receptor-like molecules exhibited a neuronal phenotype.

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