Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor‐mediated inositol 1,4,5‐triphosphate generation

Abstract

Increase in the intracellular inositol triphosphate (IP 3 ) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced a⁄nity to [125 I]Ang II (K d = 0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241^244, 250^251 and 255^256) with that of the AT1 in the mutant M8, not only increased the a⁄nity of the AT2 receptor to [125 I]Ang II (K d = 0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP 3 levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241^256 is su⁄-cient for the AT2-mediated inhibition of AT1-stimulated IP 3 generation. Moreover, these nine mutations are also su⁄cient to render the AT2 with the ability to activate phospholipase C. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…The intracellular third loop (ICL3) of the AT 2 receptor was shown to be important for its coupling to G i (Hayashida et al, 1996). Evidence of ICL3 involvement was also shown in AT 2 receptor induction of apoptotic responses in the PC12W neuronal lineage cells and in AT 2 receptor-mediated inhibition of IP 3 generation in Xenopus oocytes (Kumar et al, 2002). More recent studies implicated the involvement of G i in AT 2 receptor-dependent increases in nitric oxide synthase expression (Li et al, 2007a) and the inhibition of proximal tubule Na + -ATPase by Ang(1-7) Gohlke et al, 1998;Siragy, 2000).…”

PMA- and ANG II-induced PKC regulation of the renal Na+-HCO3−cotransporter (hkNBCe1)

“…In the renal PT, ANG II binds to the AT 1 and interacts primarily with G q proteins, leading to activation of PKC and to mobilization of Ca 2ϩ from intracellular stores (13). X. laevis oocytes lack endogenous receptors for ANG II but have powerful G q protein-linked PKC and Ca 2ϩ signaling pathways that can be activated by agonists of recombinant mammalian receptors (2,10,18,23,36). Therefore, we heterologously coexpressed the human hkNBCe1 with the rat AT 1B receptor in X. laevis oocytes.…”

RAS-Mediated Adaptive Mechanisms in Cardiovascular Tissues: Confounding Factors of RAS Blockade Therapy and Alternative Approaches

“…Activated AT2R induces a vasodilator cascade of BK/NO/cyclic guanosine monophosphate (cGMP), stimulates various protein phosphatases (SHP1, MKP1, and PP2A), and inhibits cell growth. We have shown that the AT2R exerts antagonistic effects on AT1R signaling, and this effect is mediated via the third intracellular loop of the AT2R [52]. We have also shown that the AT2R interacts with the ErbB family receptors and Na + /H + exchanger NHE6 via its third intracellular loop and C-terminal cytoplasmic domain [52,53].…”

“…We have shown that the AT2R exerts antagonistic effects on AT1R signaling, and this effect is mediated via the third intracellular loop of the AT2R [52]. We have also shown that the AT2R interacts with the ErbB family receptors and Na + /H + exchanger NHE6 via its third intracellular loop and C-terminal cytoplasmic domain [52,53]. The AT2R also interacts with a family of AT2R-interacting proteins involved in neuronal differentiation, vascular remodeling, and tumor suppression via its C-terminal domain [54].…”