Structural analysis of three peptides related to the transmambranic helix VI of AT1 receptor.
BACKGROUND
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.


OBJECTIVE
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.


METHODS
Three peptides had their conformation compared by circular dichroism (CD): L262D(259-272), L265D(259-272) (mutants) and WT(260-277).


RESULTS
Secondary structures were: beta-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). beta-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.
de Noronha
Samuel Marcos Ribeiro
SM
Ginecologia Molecular/Ginecologia, UNIFESP-R. Pedro de Toledo, 791 - 4o. Andar, V. Clementino, CEP04039032 Sao Paulo, SP, Brazil. noronha03@unifesp.br
Corrêa
Silvana Aparecida Alves
SA
Poletti
Erick Fernando
EF
Lopes
Douglas Duarte
DD
da Silva
Caroline Corrêa
CC
Sforça
Mauricio Luis
ML
Shimuta
Suma Imura
SI
Zanchin
Nilson Ivo Tonin
NI
Nakaie
Clovis Ryuichi
CR
da Silva
Ismael Dale Cotrim Guerreiro
ID
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D044140:Receptor, Angiotensin, Type 1