Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site.
Recently, we discovered a novel non-angiotensin type 1 (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 two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation 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.
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106.
Naomi J
NJ
Wangler
Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, Florida 33328.
Kira L
KL
Santos
Max-Delbrück-Center for Molecular Medicine, Berlin 13092, Germany.
Ines
I
Schadock
Proteomics Center, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642.
Fred K
FK
Hagen
Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec J1H5N4, Canada.
Emanuel
E
Escher
Max-Delbrück-Center for Molecular Medicine, Berlin 13092, Germany.
Michael
M
Bader
Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, Florida 33328; Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, Florida 32611.
Robert C
RC
Speth
Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106; Vascular Drug Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106. Electronic address: vardan.karamyan@ttuhsc.edu.
Vardan T
VT
Karamyan
D000809:Angiotensins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D011247:Pregnancy; D016548:Prosencephalon / Q000166:cytology; D011485:Protein Binding
D000809:Angiotensins; D008666:Metalloendopeptidases; C062316:neurolysin