A Conserved NPLFY Sequence Contributes to Agonist Binding and Signal Transduction but Is Not an Internalization Signal for the Type 1 Angiotensin II Receptor (*)

Abstract

A conserved NPXY sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn and was reduced by replacement of Pro or Tyr. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar¹,Ile⁸]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe, Tyr, and Phe, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe residue contributes significantly to agonist binding, and Asn is required for normal receptor activation and signal transduction.