Effects of mutations in the highly conserved DRY motif on binding affinity, expression, and G-protein recruitment of the human angiotensin II type-2 receptor

The signaling pathways for the seven transmembrane G-protein coupled angiotensin II receptors (AT₁ and AT₂) are just beginning to be understood. While these receptors play an important role in the development and differentiation of many tissues, including the cardiovascular and central nervous systems, information about amino acid motifs involved in angiotensin II-mediated signaling is only available for the AT₁ receptor subtype. In the present study, we mutated the conserved DRY^141-143 motif in the AT₂ receptor, which is thought to be involved in G-protein recruitment. Expression of wild type and mutant receptors in CHO-K1 cell plasma membranes was confirmed using radioligand binding analyses. Our findings indicate a significant change in the binding affinities (kD) and capacities (B_max) of the mutant receptors relative to wild type. Alanine substitutions of D¹⁴¹ and DRY^141-143 resulted in a significant decrease of binding affinity for both Sar¹Ile⁸-angiotensin II (SarIle-Ang II) (mixed agonist/antagonist) and angiotensin II (agonist). The binding affinities following alanine substitutions of R¹⁴² and Y¹⁴³ were not significantly different from wild type receptor. Interestingly, the R¹⁴²-A and Y¹⁴³-A mutants revealed a significant decrease in binding levels from wild type with SarIle-Ang II, but not angiotensin II. The effect of GTPγS on angiotensin II binding affinity between wild type and mutant receptors was similarly significant. The D¹⁴¹-A, Y¹⁴³-A, and DRY^141-143-AAA mutant receptors showed a marked decrease in GTPγS-induced angiotensin II affinity shift. The R¹⁴²-A GTPγS binding affinity shift was not different from the wild type receptor. Our results support the hypothesis that the DRY motif plays a significant role in the binding affinity, structural stability and G-protein recruiting of the AT₂ receptor.