A previously unappreciated deubiquitinase activity of MCP-induced protein 1 contributes to its role in dampening inflammatory signaling.

In the current model of receptor activation, the given hormone is not involved in the conversion of the inactive receptor (R) to the fully active state (R*). Rather, it preferentially selects the activated receptor conformation, thereby shifting the equilibrium toward R*. The hormone angiotensin II (Ang II) contains two residues, Tyr4 and Phe8, that are essential for agonism. We show that the conserved Asn111 in transmembrane helix III of the AT1 angiotensin receptor directly interacts with the Tyr4 side chain. A decrease in the size of the Asn111 side chain induces an intermediate activated receptor conformation (R'). The Ang II analogue [Sar1,Ile4,Ile8]Ang II fully activates the N111G mutant, indicating that either the transition from R' to R* or the stabilization of the R* state requires binding by Ang II but not its Tyr4 and Phe8 side chains. In contrast, [Sar1,Ile4,Ile8]Ang II binds to but does not activate the wild-type AT1 receptor (R), suggesting that in the wild-type receptor spontaneous occurrence of R' and R* states is rare. Thus, Ang II through interactions involving Tyr4 and Phe8 induces a transition from R to R' and through unspecified interactions induces transition from R' to R* states rather than stabilizing the spontaneously generated R* state by "conformational, selection".

To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT1 receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys102 and Arg167 mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar1,Ile8]angiotensin II, and several carboxylate analogues of losartan. An Ala199 substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan. Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys199 is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT1 receptor involves multiple contacts with residues such as Lys199 and His256 that constitute the same subsite of the ligand binding pocket. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.

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The structural model of AT1 angiotensin receptor contains seven-transmembrane alpha-helices with three interhelical loops on either side of the membrane. The angiotensin II binding pocket within the receptor is not clearly defined. We showed earlier that Lys199 in transmembrane-helix-5 of the AT1 receptor binds the COOH-terminal alpha-carboxyl group of angiotensin II (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 2284-2289). We now show that His183 and Asp281, both located in the extracellular domain of the AT1 receptor, are involved in binding the NH2-terminal Asp1 and Arg2 residues of angiotensin II, respectively. The Asp1/His183 interaction appears to be weak and is unlikely to be important for agonism. But the loss of Arg2/Asp281 interaction leads to partial agonism of the receptor. The action of non-peptide agonists is not affected by Asp281 mutations. These results suggest that several independent interactions between angiotensin II and AT1 receptor are necessary for full agonism. Since L-162,313 the non-peptide agonist of the AT1 receptor is a partial agonist that does not make contact with Asp281, we speculate that the degree of agonism may be increased if it is redesigned to make contacts with Asp281.