Identification of Membrane-bound Variant of Endopeptidase 24.16 as the non-AT1, non-AT2Angiotensin II Binding Site

Objective. To determine the identity of the non-AT1, non-AT2 angiotensin II (AngII) binding site in the mammalian brain. Background. New discoveries about the renin-angiotensin system continue to abound in the 21st century, e.g., discovery of a renin receptor, identification of the mas oncogene protein as the angiotensin 1-7 receptor, discovery of an enzyme (angiotensin-converting enzyme-2, ACE-2) that converts AngII to angiotensin 1-7, use of AT2 receptor agonists as therapeutic agents, and discovery of a novel non-AT1, non-AT2 angiotensin binding site in the brain. Methods. An angiotensin analog, photoaffinity probe 125I-sarcosine1,benzoylphenylalanine8-AngII was used to specifically label the non-AT1, non-AT2 AngII binding site in membranes prepared from mouse forebrain and HEK293 cell overexpressing mouse neurolysin in the presence of AT1 receptor-saturating concentrations of losartan, AT2 receptor-saturating concentrations of PD123319, and 150 μM parachloromercuribenzoate. The 125Isarcosine1, benzoylphenylalanine8-AngII radiolabeled binding site was purified by 2-Dimensional electrophoresis and analyzed by mass spectrometry, or solubilized and immunoprecipitated. Saturation binding assays or in vitro autoradiography with 125I-sarcosine1,isoleucine8-AngII measured expression of the binding site in brain and HEK293 cell membranes. Results. The binding site is a ~75kDa membrane protein that fits a mass spectrometric profile of neurolysin. An antibody to neurolysin precipitated the 125Isarcosine1, benzoylphenylalanine8-AngII labeled protein. Overexpression of neurolysin in HEK293 cells increased non-AT1, non-AT2 125I-sarcosine1,isoleucine8-AngII binding. Binding of 125I-sarcosine1, isoleucine8-AngII to the non-AT1, non-AT2 binding site in neurolysin knock-out mouse brains was dramatically decreased compared to wild-type brains. Conclusion. A membrane-bound variant of the metalloendopeptidase neurolysin (E.C.3.4.24.16) is the novel AngII binding protein. Grants. Supported by NHLBI HL-096357.