Objective: This study was designed to demonstrate that the principle of molecular recognition underlying high-affinity binding of angiotensin II to the type 2 (AT2) receptor is distinct from that of the type 1 (AT1) receptor. In general, the same functional pharmacophores in hormones are used to bind and activate different subtypes of cell surface receptors. However, the binding of angiotensin II to the AT2 receptor is distinct from that of the AT1 receptor.
Design and methods: To systematically evaluate the effect of modification of angiotensin II side chains on binding to both the receptors, several analogs of angiotensin II were synthesized. Rat AT1 or AT2 receptors expressed in COS1 cell membranes were used to determine the affinity of analogs using radioligand competition binding experiments under equilibrium conditions.
Results: Modifications of all angiotensin II side chains affected binding to the AT2 receptor to nearly similar extents. In contrast, binding to the AT1 receptor was significantly affected by modifications at side chain positions 2, 4, 6 and 7. In accordance with previous observations that Tyr4- or Phe8-modified angiotensin II analogs antagonized vasoconstriction mediated exclusively by the AT1 receptor, binding to the AT1 receptor was significantly dependent on Tyr4 or Phe8 of angiotensin II whereas binding to the AT2 receptor was not. Rather surprisingly, the affinity profile of several angiotensin II analogs towards the AT2 receptor was similar to the measured affinity of the constitutively active N111G mutant AT1 receptor.
Conclusions: These results suggest that the AT2-receptor pharmacophore is very distinct from that of the AT1 receptor. The AT1 receptor is in a constrained conformation and is activated only when bound to angiotensin II. In contrast, the AT2 receptor is 'relaxed' in that no single interaction is critical for binding, like the N111G mutant AT1 receptor, which is constitutively active.