Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop
- 1 October 1989
- journal article
- research article
- Published by Springer Nature in The Journal of Membrane Biology
- Vol. 111 (2) , 141-153
- https://doi.org/10.1007/bf01871778
Abstract
We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung β2-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 β2-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 as125I-cyanopindolol tabeled β2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the125I-cyanopindolol binding domain of the β2-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 β2-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 β2-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 β2-adrenergic receptor-like molecules exhibited a neuronal phenotype.This publication has 50 references indexed in Scilit:
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