Received for publication February 3, 2005.
Revised May 13, 2005.
Accepted for publication May 17, 2005.

Single Mutations at Asn²⁹⁵ and Leu³⁰⁵ in the Cytoplasmic Half of TM7 of the AT₁ Receptor Induce Promiscuous Agonist Specificity for Angiotensin II Fragments - A PSEUDO-CONSTITUTIVE ACTIVITY

Abstract

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). Herein we show 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 IP 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 due to lack of endogenous Ang II fragments produced by the cells - a pseudo-constitutive activity. Surprisingly, the Ang II analog, [Sar¹,Ile⁴,Ile⁸]Ang II, and the native angiotensin II fragments Ang 1-7, Ang IV, and Ang 5-8 that 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 (RN). These data have identified 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.

Key words: Angiotensin, IP3/DAG, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Structure/function/mechanism

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