Single mutations at Asn²⁹⁵ and Leu³⁰⁵ in the cytoplasmic half of transmembrane α-helix domain 7 of the AT₁ receptor induce promiscuous agonist specificity for angiotensin II fragments: A pseudo-constitutive activity

The most striking feature of a G protein-coupled receptor (GPCR) is its highly exclusive agonist specificity. This feature guarantees that a GPCR recognizes only its specific native agonist(s). In this study, we showed that two point mutations of N295S and L305Q enabled the AT₁ receptors to recognize multiple Ang II fragments. Similar to the well established constitutively active AT₁ mutant receptor N111G, the mutations of N295S and L305Q induced an increased production of basal inositol 1,4,5-phosphates in the absence of exogenous Ang II when expressed in HEK293 cells. Distinct from the N111G, however, is the fact that the increased basal activity disappeared in COS-7 cells because of the lack of endogenous Ang II fragments produced by the cells - a pseudo-constitutive activity. It is surprising that the Ang II analog [Sar¹,Ile⁴,Ile ⁸]Ang II and the native angiotensin II fragments Ang 1-7, Ang IV, and Ang 5-8, which are inactive in activating the wild-type receptor, activated N295S and L305Q. Results generated by lowering the Na⁺ concentration suggest that the mutant N295S and L305Q may be trapped in neutral conformational states (R^N). These data allow us to identify for the first time a novel pattern of GPCR mutations with a broad spectrum of agonist specificity, suggesting possible existence of functional GPCRs in nature that are activated through conformational "selection" rather than "induction" mechanisms.