C. Bihoreau et al., MUTATION OF ASP(74) OF THE RAT ANGIOTENSIN-II RECEPTOR CONFERS CHANGES IN ANTAGONIST AFFINITIES AND ABOLISHES G-PROTEIN COUPLING, Proceedings of the National Academy of Sciences of the United Statesof America, 90(11), 1993, pp. 5133-5137
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 beta2-adrenerg
ic and luteinizing hormone receptors. To determine whether this aspart
ic acid is also involved in the function of the rat vascular angiotens
in II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagi
ne or by glutamic acid. When expressed in CHO cells, the two mutants a
nd the wild-type receptor displayed similarly high affinities for the
agonist [Sar1, Tyr(I-125)4]angiotensin II [where Sar is sarcosine and
Tyr(I-125) is monoiodinated tyrosine] and the other agonists: ([Sar1]a
ngiotensin II > angiotensin II > angiotensin III >> angiotensin I). Ho
wever, the Asn74 mutant shows striking differences in its affinity for
some antagonists when compared with the wild-type receptor: the affin
ity for DUP753 was decreased 10-fold, whereas it was increased 6-fold
for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmac
ological changes were associated with a major defect in transmembrane
signaling, since angiotensin II was unable to stimulate inositol phosp
hate 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 wa
s able to promote DNA synthesis through the wild type but not through
the mutated receptors. These data indicate that the conserved Asp74 re
sidue of the AT1a receptor is important for the binding of angiotensin
II antagonists and is essential for the transmembrane signaling casca
de.