The Conformational Change Responsible for AT1 Receptor Activation Is Dependent upon Two Juxtaposed Asparagine Residues on Transmembrane Helices III and VII

Abstract

A model of the angiotensin AT 1 receptor and site-directed mutagenesis were used to identify key residues involved in ligand binding. We propose that substitution of these residues causes the loss of an interaction between transmembrane helices III and VII, which allows the AT 1 receptor to "relax" into its active conformation.The renin-angiotensin system plays a vital role in the regulation of cardiovascular function, and its activity may be abnormal in a number of disease states. The effector molecule of this system, the octapeptide angiotensin II (Ang II), 1 has a number of actions in a variety of cell types (1). Two distinct Ang II receptor subtypes, AT 1 and AT 2 have been identified (2). This characterization is based on the differential selectivity for these two receptors of the non-peptide Ang II receptor antagonists, DuP 753 (losartan; AT 1 selective) and PD123177 (AT 2 selective). In addition, the modified peptides CGP42112A (3) and [p-amino-Phe 6 ]angiotensin II (pNH 2 F 6 AII) (4), both display selectivity for the AT 2 receptor over the AT 1 receptor. Furthermore, several studies suggest that AT 1 and AT 2 receptors display subtle differences in their binding affinities for Ang II and angiotensin III (Ang III). AT 2 receptors bind Ang II and Ang III with equal affinity (5-8), while AT 1 receptors exhibit a slightly higher affinity for Ang II, between 3-and 20-fold greater than Ang III (9 -11). Many of the recognized actions of Ang II appear to be mediated through AT 1 receptors, predominantly through activation of the phospholipase C signal transduction pathway.AT 1 receptors have been cloned from a variety of species and tissues (9 -15). All these AT 1 receptors consist of a single polypeptide, 359 amino acids in length, arranged with a topography comprising seven ␣-helical transmembrane regions (TM I-VII), typical of the G-protein-coupled receptor family. They display a high degree of sequence identity at the amino acid level (over 94% identical between all mammalian species). Two closely related isoforms (AT 1A and AT 1B ) encoded by different genes have been identified in the rat and mouse (13, 16) Recently AT 2 receptors have been cloned (17-19), which are also members of the G-protein-coupled receptor family. They consist of 363 amino acids and share only 34% sequence identity at the amino acid level to the AT 1 receptors.Mutagenesis studies of the AT 1 receptor have recently identified residues important in the binding of the non-peptide antagonist, losartan, and the natural ligand Ang II. These residues appear to be mutually exclusive, since amino acid substitutions, which affect losartan binding, do not alter Ang II binding, and vice versa. The major determinants of losartan binding appear to be residues located within the transmembrane helices . In contrast, the major determinants of Ang II binding appear to be residues present in the extracellular regions, particularly in the N-terminal extension adjacent to the first transmembrane helix and in the C-terminal part of the third extracellula...

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO‐K1 cells expressing human angiotensin II AT1 receptors

“…This, in turn, triggers cell growth and constriction (Vincentini & Villereal, 1986). The initial AT 1 receptor-evoked responses can conveniently be measured in primary cell cultures and in cell lines (Dickinson et al, 1994;Koh et al, 1994;Panek et al, 1995;Perlman et al, 1995;Balmforth et al, 1997) and appear to be common to the biological actions of angiotensin II. In addition, it was also observed by Dickinson et al (1994) that angiotensin II increases the rate at which rabbit aortic smooth muscle cells acidify their environment with products of their energy metabolism.…”

The angiotensin II type 1 receptor and receptor-associated proteins

“…B a s e d o n a n u m b e r o f pharmacological, mutagenesis and computer-oriented modeling studies [63][64][65][66][67][68][69][70][71][72][73][74][75][76], the general agreement is that a positively-charged residue of Lys199 on transmembrane domain (TM)-5 of the AT1 receptor interacts with the -carboxyl group of Phe8 in Ang II. This interaction is crucial for high affinity binding of Ang II to the receptor.…”

Biochemical properties of renin and prorenin binding to the (pro)renin receptor

“…67 It has been suggested that a specific interaction between the third and the seventh transmembrane helixes may be a major determinant of AT 1 receptor isomerization from an inactive to active conformation. 68 Binding of a5b1 to urokinase-type plasminogen activator receptors causes a change in the conformation to block the functions of this complex by short b1-chain peptides without affecting a5b1-mediated fibronectin binding. 69 Therefore, the decoy peptide blunted the interaction of renin with (P)RR possibly by changing specific space within the receptor.…”