125 I-Angiotensin 1–7 binds to a different site than angiotensin 1–7 in tissue membrane preparations

Purpose To study the receptor for Angiotensin (Ang) 1–7 using a radioligand ( 125 I-Ang 1–7)-binding assay. For more than a decade, Mas has been viewed as the receptor for Ang 1–7; however, Ang 1–7 binding has not been pharmacologically characterized in tissue membrane preparations. Methods Radioligand-binding assays were carried out using tissue membrane preparations using radioiodinated Angiotensin 1–7 ( 125 I-Ang 1–7) to characterize its binding site. Non-radioactive 127 I-Ang 1–7 was used to test if the addition of an iodine to the tyrosine 4 moiety of Ang 1–7 changes the ability of Ang 1–7 to competitively inhibit 125 I-Ang 1–7 binding. Results 125 I-Ang 1–7 binds saturably, with moderately high affinity (10–20 nM) to a binding site in rat liver membranes that is displaceable by 127 I-Ang 1–7 at nanomolar concentrations (IC 50 = 62 nM) while Ang 1–7 displaces at micromolar concentrations (IC 50 = 80 µM) at ~22 °C. This binding was also displaceable by inhibitors of metalloproteases at room temperature. This suggests that 125 I-Ang 1–7 binds to MMPs and/or ADAMs as well as other liver membrane elements at ~ 22 °C. However, when 125 I-Ang 1–7-binding assays were run at 0–4 °C, the same MMP inhibitors did not effectively compete for 125 I-Ang 1–7. Conclusions The addition of an iodine molecule to the tyrosine in position 4 of Ang 1–7 drastically changes the binding characteristics of this peptide making it unsuitable for characterization of Ang 1–7 receptors.