Photolabeling, purification and identification of the non‐AT 1 , non‐AT 2 angiotensin binding site

The recently discovered non‐AT 1 , non‐AT 2 angiotensin binding site is hypothesized to be a new member of the renin‐angiotensin system. However, its identity remains unknown. To isolate and identify this protein, neonatal (P10) mouse forebrain homogenates were incubated with AT 1 (ZD7155) & AT 2 (PD123319) receptor antagonists, p‐chloromercuribenzoate and photoreactive ligand 125 I‐SBpa Ang II (± Ang II to specifically identify the binding site). After exposure to UV light, membrane suspensions were pelleted and subjected to SDS/PAGE. Gel lanes were cut into 3mm segments and assayed for 125 I. Specific binding of photolyzed radioligand was seen at 75kDa. Proteins extracted from this region were focused on an immobilized pH gradient strip. Migration of 125 I was monitored in the strip. The radioactive segment (pI ≈ pH 5.5–6.5) was cut out and proteins were extracted. The protein extract was resolved by 2D gel electrophoresis and revealed by autoradiography. The region corresponding to the photolabeled protein was excised and analyzed by mass spectroscopy. LC‐MS/MS analysis identified ~10 candidate proteins with 9 – 27 peptide matches per protein identification. The ability of these candidate proteins to interact with angiotensins is being evaluated to identify this novel binding site. Supported by NHLBI HL‐096357, American Radiolabeled Chemicals & TTUHSC SOP start up funds.