Interactions between conserved residues in transmembrane helices 2 and 7 during angiotensin AT1 receptor activation.
Site-directed mutagenesis studies and independent molecular modeling studies were combined to investigate the network of inter-residue interactions within the transmembrane region of the angiotensin AT(1a) receptor. Site-directed mutagenesis was focused on residues Tyr292, Asn294, Asn295, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT(1a) receptor activation. Replacement of Tyr292 by aspartate in helix 7 abolished radioligand binding to both Y292D and D74Y/Y292D mutant receptors. Reciprocal mutations of Asp74/Asn294, Ser115/Asn294, Ser252/Asn294, and Asn298/Sen115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT(1a) receptor activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the TM region of the AT(1) receptor and the mutants as well as of their complexes with angiotensin II, which were used to rationalize the possible reasons of impairing of function of some mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct strong interaction in the resting conformation of the AT(1a) receptor. Balance of interactions between residues in helix 2 (as D74) and helix 7 (as N294, N295 and N298) in the AT(1) receptors, however, has a crucial role both in determining their functional activity and levels of their expression.
Nikiforovich
Gregory V
GV
Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St Louis, MO 63110, USA. gregory@ccb.wustl.edu
Zhang
Meng
M
Yang
Qing
Q
Jagadeesh
Gowraganahalli
G
Chen
Hao-Chia
HC
Hunyady
László
L
Marshall
Garland R
GR
Catt
Kevin J
KJ
D000596:Amino Acids; D000818:Animals; D017124:Conserved Sequence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D044140:Receptor, Angiotensin, Type 1