Preliminary biochemical characterization of the novel, non-AT1, non-AT2 angiotensin binding site from the rat brain

Abstract

A novel binding site for angiotensins II and III was recently discovered in brain membranes in the presence of the sulfhydryl reactive angiotensinase inhibitor parachloromercuribenzoate. This binding site is distinctly different from the other known receptors for angiotensins: AT₁, AT₂, AT₄, and mas oncogene protein (Ang 1-7 receptor). Preliminary biochemical characterization studies have been done on this protein by crosslinking it with (125)I-labeled photoaffinity probes and solubilizing the radiolabeled binding site. Polyacrylamide gel electrophoresis studies and isoelectric focusing indicate that this membrane bound binding site is a protein with a molecular weight of 70-85 kDa and an isoelectric point of ~7. Cyanogen bromide hydrolysis of the protein yielded two radiolabeled fragments of 12.5 and 25 kDa. The protein does not appear to be N-glycosylated based upon the failure of PNGaseF to alter its migration rate on a 7.5% polyacrylamide gel. The binding of angiotensin II to this protein is not affected by GTPγS or Gpp(NH)p, suggesting that it is not a G protein-coupled receptor. Further characterization studies are directed to identify this protein either as a novel angiotensin receptor, an angiotensin scavenger (clearance receptor) or an angiotensinase.

Fig. 1

High affinity binding of ¹²⁵I-azido-Ang II, ¹²⁵I-SBpa-Ang II, and ¹²⁵I-SI-Ang II to the novel angiotensin binding site in rat brain membranes. a Representative saturation analyses of ¹²⁵I-SI-Ang II (B_max = 2.6 ± 0.2 fmol/mg wet wt, K_d = 1.35 ± 0.3 nM) and ¹²⁵I-azido-Ang II (B_max = 2.24 ± 0.3 fmol/mg wet wt, K_d = 2.7 ± 0.78 nM) binding in rat cerebral cortical membranes in the presence of 10 μM PD123319, 10 μM losartan, and 0.3 mM PCMB (1 h incubation at 24°C). b Representative saturation analyses of ¹²⁵I-SI-Ang II (B_max = 2.77 ± 0.15 fmol/mg wet wt, K_d = 1.4 ± 0.2 nM) and ¹²⁵I-SBpa-Ang II (B_max = 1.75 ± 0.1 fmol/mg wet wt, K_d = 0.6 ± 0.1 nM) binding in rat forebrain membranes in the presence of 10 μM PD123319, 10 μM losartan, and 0.3 mM PCMB (1 h incubation at 24°C); n = 3 for each assay

Fig. 2

Radiolabeling of the novel angiotensin binding site with photoaffinity probe ¹²⁵I-azido-Ang II in rat forebrain membranes (representative results from four independent experiments). Left SDS-PAGE (12%) analysis of radio-photolabeled rat forebrain membranes followed by Coomassie blue staining; bracketed areas represent a segment of the lanes (between molecular masses 86 and 50 kDa) corresponding to the highest specific binding (10 μM Ang II displaceable) of the radio-photoligand (“Total”—binding of ¹²⁵I-azido-Ang II to the membranes in the presence of 10 μM losartan, 10 μM PD123319, and 0.3 mM PCMB; “Non-specific”—binding of ¹²⁵I-azido-Ang II under the same experimental conditions plus 10 μM Ang II). Right migration of ¹²⁵iodine in the “Total” and “Non-specific” lanes of the same SDS gel (0.5 cm sections)

Fig. 3

Analysis of N-glycosylation status of the novel angiotensin binding site (representative results from three independent experiments). Left SDS-PAGE (7.5%) analysis and Coomassie blue staining of radio-photolabeled (¹²⁵I-SBpa-AngII), semipurified novel angiotensin binding site without (BS) and with (BS + PNGaseF) PNGaseF treatment. Glycoprotein fetuin was used as a positive control in this experiment. Right migration of ¹²⁵iodine in the “BS” and “BS + PNGase” lanes of the same SDS gel (~0.3 cm sections)

Fig. 4

Representative autoradiogram of Tris-tricine-SDS-PAGE (16.5%) analysis of CNBr and PNGaseF digests of radio-photolabeled (¹²⁵I-SBpa-AngII) and semipurified novel angiotensin binding site. Lane 1 contains free/unbound 125I-SBpa-AngII, lane 2 contains untreated binding site, lane 3 contains PNGaseF-treated binding site, lane 4 contains CNBr-treated binding site, lane 5 contains CNBr and PNGaseF-treated binding site

Fig. 5

Competition by Ang II for ¹²⁵I-SI Ang II (~0.25 nM) binding to the AT1 angiotensin receptor (AT1R; in liver membranes) or the novel angiotensin binding site (non-AT1/AT2; in cerebral cortical membranes) in the presence and absence of 50 μM GTPγS (a) or 50 μM Gpp(NH)p (b); n = 3