We have previously shown that individual β-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that β-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel β-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace 125I-Sar1Ile8 Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > β-Pro7 Ang III=Ang II > β-Tyr4 Ang III ≥ PD123319 >> β-Phe8 Ang III >> β Arg2 Ang III=β-Val3 Ang III >> β-Ile5 Ang III. The novel analogue β-Pro7 Ang III was the most selective AT2R ligand tested, which was >20 000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that β-Pro7 Ang III was an AT2R agonist, we compared β-Pro7 Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), β-Pro7 Ang III had no effect. During low-level AT1R blockade, both Ang III and β-Pro7 Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, β-Pro7 Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

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