Mutation of Asp74of the Rat Angiotensin II Receptor Confers Changes in Antagonist Affinities and Abolishes G-Protein Coupling

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in β2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involve...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1993-06, Vol.90 (11), p.5133-5137
Main Authors: Bihoreau, Claire, Monnot, Catherine, Davies, Eleanor, Teutsch, Betty, Bernstein, Kenneth E., Corvol, Pierre, Clauser, Eric
Format: Article
Language:English
Subjects:
Agonists
Amino acids
Biochemistry
Cell lines
Cells
CHO cells
COS cells
Genetic mutation
Ligands
Receptors
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Summary:Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in β2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II receptor subtype 1 (AT1a), Asp74was replaced either by asparagine or by glutamic acid. When expressed in CHO cells, the two mutants and the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(125I)4]angiotensin II [where Sar is sarcosine and Tyr(125I) is monoiodinated tyrosine] and the other agonists: ([Sar1]angiotensin II > angiotensin II > angiotensin III >> angiotensin I). However, the Asn74mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affinity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmacological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosphate production and increase cytosolic Ca2+concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II was able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74residue of the AT1areceptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling cascade.
ISSN:0027-8424