Effect of cysteine 18 substitution for serine on Angiotensin II AT1 receptor on structure-activity relationship and intracellular expression

• Grant number: 06/00295-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2006
• End date: December 31, 2006
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Suma Imura Shimuta
• Grantee: Renan Paulo Martin
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

A series of site-directed mutagenesis of recombinantly expressed AT1 receptor combined with the modification of AII have led to the identification of several contacts between AII and its receptor, important for biochemical and pharmacological characterization. Previous studies have provided evidence that S-S bridge established between Cys101 and Cys180 is crucial for the binding and activation of AT1 receptor but few data are available about the second S-S bond between Cys18 and Cys274. The objective of this study is to better define the function of the second S-S bond through the C18S mutant, which through the substitution of Cys18 with Ser, eliminates the second salt bridge. We’ll investigate the binding affinity of this mutant to the ligand angiotensin II and its analogs. Heterolog competition binding assay will be performed using 3H-AII and unlabeled AII analogs bearing substitution on N-terminal segment, such as Sar1-AII, Lys2-AII and Sar1Lys2-AII. In addition, the intracellular localization of the receptor will be evaluated between two hypotheses: a) incorrect protein folding, leading to a deficient maturation process; b) constitutive receptor internalization. To test the first hypothesis, immunocytochemical assay with calnexin will be performed, in order to examine if calnexin, a molecular chaperone, is involved in the structure of glycosylated receptor protein while crossing the endoplasmatic reticulum. To test the second hypothesis, specific antagonists of AT1 receptor will be used as inverse agonists to induce receptor externalization, and thus inhibiting the stimulated basal activity of agonist-independent IP3 formation. (AU)