Characterization of a binding site for angiotensin IV on bovine aortic endothelial cells

We have characterized a specific binding site for angiotensin IV on bovine aortic endothelial cell membranes. Pseudo-equilibrium studies at 37°C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (K_d=0.71; average of two experiments that yielded values of 0.71 and 0.72 nM). The binding site is saturable and relatively abundant with a maximal binding capacity of 0.59 pmol/mg protein (average of two experiments that yielded values of 0.39 and 0.78 pmol/mg of protein). Non-equilibrium kinetic analyses at 37°C revealed a calculated K_d of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site is pharmacologically distinct from the classic angiontensin receptors AT₁ or AT₂. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar¹, Val⁵, Ala⁸]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2′-(H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl)methyl]-3H-imidazo [4,5-β]pyridine H₂O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacety)4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). A few unrelated hormones (bradykinin, [Arg⁸]vasopressin, endothelin-1, atrial natriuretic factor, isoproterenol and adrenocorticotropic hormone) were unable to inhibit any ¹²⁵I-angiotensin IV binding. The affinities of different structural analogues of angiotensin IV revealed that the N-terminal position is critical for receptor recognition and the C-terminal proline is also important. GTPγS and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg²⁺ and Ca²⁺ were shown to diminish the binding of ¹²⁵I-angiotensin IV. Cross-linking of ¹²⁵I-angiotensin IV to bovine aortic endothelial cell membranes in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a major band of 186±12 kDa. The presence in high concentration of this angiotensin binding site an aortic endothelial cells suggests the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.