Structural analysis of three peptides related to the transmambranic helix VI of AT1 receptor☆☆☆
published online 14 December 2009.
Abstract
Introduction
Angiotensin II (AII) is the main active product of the renin angiotensin system. Better known effects of AII are via AT1 receptor (AT1R). Expression of AT1R mutants (L265D and L262D) in CHO cells increased cAMP formation when compared to CHO cells expressing the wild type (WT) AT1R. Morphological transformation of CHO cells transfected with mutants correlated with their increased cAMP formation. DNA synthesis was inhibited in these cells too, indicating that cAMP promotes inhibitory effects on transfected CHO cells growth and causes their morphological change from a tumorigenic phenotype to a non-tumorigenic one.
Objectives
To assess the importance of leucine 262 and 265 in determining AT1R structure by means of a comparative structural analysis of two mutant peptides and of a wild-type fragment.
Methodology
Three peptides had their conformation compared by circular dichroism (CD): L262D259–272, L265D259–272 (mutants) and WT260–277.
Results
Secondary structures were: β-turn for WT and L262D and random coil for L265D.
Conclusions
Strong correlation was found in the results of biochemical, cellular and structural approaches used to compare WT AT1R to mutant types. Random coil structure of the L265D mutant may be a key point to explain those changes observed in biochemical (binding and signal transduction) and proliferation assays (Correa et al., 2005). β-Turn formation is an important step during early protein folding and this secondary simple structure is present in L262D and WT, but not in L265D. Therefore, leucine 265 seems to play a crucial role in determining an entirely functional AT1R.
aGinecologia Molecular/Ginecologia, UNIFESP-R. Pedro de Toledo, 791 – 4o. Andar, V. Clementino, CEP04039032 Sao Paulo, SP, Brazil
bDep. Biofisica-R. Botucatu, 840 – 7o. Andar, V. Clementino, Sao Paulo, SP, Brazil
cLaboratorio Nacional de Luz Sincroton-Rua Giuseppe Maximo Scolfaro, 10000-Polo II de Alta Tecnologia de Campinas-Campinas, SP, Brazil
Corresponding author. Address: Molecular Gynecology Laboratory, Gynecology Department, Federal University of Sao Paulo, Rua Pedro de Toledo, 781 – 4th floor, Sao Paulo, 04039-032 SP, Brazil. Tel.: +55 55791534; fax: +55 11 55793321.
☆ This manuscript is related to the The Fifth International Symposium on Peptide Receptors and Kinin 2009.
☆☆ Supported by Grants 2005/54160–2 and 2008/50776-7 from the Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)-Brazil.
ABSTRACT