H-His-Ala-OH

The phenolic side chain of Tyr(4) present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, (125)I-Ang II and [(125)I-Sar(1)-Ile(8)]Ang II, as well as the AT2 receptor-specific peptidic ligand (125)I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of (125)I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor.

1. During the course of studies directed to determine the transport of Angiotensin II AT(2) receptors in the rat brain, we found that stab wounds to the brain revealed a binding site recognized by the AT(2) receptor ligand CGP42112 but not by Angiotensin II. 2. We localized this novel site to macrophages/microglia associated with physical or chemical injuries of the brain. 3. The non-Angiotensin II site was also highly localized to inflammatory lesions of peripheral arteries. 4. In rodent tissues, high binding expression was limited to the spleen and to circulating monocytes. A high-affinity binding site was also characterized in human monocytes. 5. Lack of affinity for many ligands binding to known macrophage receptors indicated the possibility that the non-Angiotensin II CGP42112 binding corresponds to a novel site.6. CGP42112 enhanced cell attachment to fibronectin and collagen and metalloproteinase-9 secretion from human monocytes incubated in serum-free medium but did not promote cytokine secretion. 7. When added in the presence of lipopolysaccharide, CGP42112 reduced the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines TNF-alpha, IL-1, IL-1 beta, and IL-6, and increased protein kinase A. 8. Molecular modeling revealed that a CGP42112 derivative was selective for the novel macrophage site and did not recognize the Angiotensin II AT(2) receptor. 9. These results demonstrate that CGP42112, previously considered as a selective Angiotensin II AT(2) ligand, recognizes an additional non-Angiotensin II site different from AT(2) receptors. 10. Our observations indicate that CGP42112 or related molecules could be considered of interest as potential anti-inflammatory compounds.

Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [125I-Sar1-Ile8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [125I-Sar1-Ile8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.