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Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-AT1, non-AT2 angiotensin binding site

Item Type:Article
Title:Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-AT1, non-AT2 angiotensin binding site
Creators Name:Wangler, N.J. and Santos, K.L. and Schadock, I. and Hagen, F.K. and Escher, E. and Bader, M. and Speth, R.C. and Karamyan, V.T.
Abstract:Recently, we discovered a novel non-AT1, non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to 2-D gel electrophoresis followed by mass spectrometry identification of proteins in the 2-D gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated (IP) after photoradiolabeling. IP studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins.
Keywords:Angiotensin, Cardiovascular, Hormone Receptors, Ligand Binding Protein, Pharmacology, Photoaffinity Labeling, Radioreceptor Assays, Renin Angiotensin System, Neurolysin, Oligopeptidase, Animals, Mice
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:287
Number:1
Page Range:114-122
Date:2 January 2012
Official Publication:https://doi.org/10.1074/jbc.M111.273052
PubMed:View item in PubMed

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