Mohiuddin Ahmed Bhuiyan

Binding sites of valsartan, candesartan and losartan with angiotensin II receptor 1 subtype by molecular modeling

AIMS This study was designed to examine the importance of interaction in the binding of selective angiotensin II receptor antagonists to angiotensin II type 1 receptor using molecular modeling. The AT(1) antagonists used in this study were valsartan, candesartan and losartan. MAIN METHODS AT(1) receptor structural model was constructed by homology modeling using structural models of rhodopsin photointermediates. Through molecular modeling, possible binding sites for these drugs were suggested to lie between transmembrane domains (TM) 3, 5, and 6 of AT(1) receptor. KEY FINDINGS The carboxylic acid group and tetrazole ring of valsartan possibly interact with Lys199 of TM5 and Ser109 of TM3 and Asn295 of TM7 of AT(1) receptor, respectively. In candesartan, carboxylic group, tetrazole ring, and ethoxy group oxygen possibly interact with Lys199 of TM5, Ser109 of TM3 and Asn295 of TM7 and Gln257 of TM6, respectively. In losartan, tetrazole ring and hydroxymethyl group possibly interact with Asn295 of TM7 and Ser109 of TM3, respectively. SIGNIFICANCE The results of the present study suggested that candesartan interacts at a higher number of binding sites compared to valsartan whereas losartan has a lower number of binding sites with the amino acid residues of the AT(1) receptor. These findings are consistent with the data of the radioligand-binding studies of the antagonists with the AT(1) receptor.

Mutagenesis of important amino acid reveals unconventional homologous internalization of β1-adrenergic receptor

AIMS The study was designed to examine the internalization of Asp104Lys mutant of beta(1)-adrenergic receptor (beta(1)-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (betaARK1) and beta-arrestin1 on this internalization of Asp104Lys mutant of beta(1)-AR. MAIN METHODS Binding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of beta(1)-ARs stably expressed in HEK 293 cells as assessed by [(3)H] CGP12177 radioligand. We have performed GRK2 and beta-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of beta-arrestin1 gene. KEY FINDINGS In the present study, the binding affinity of (-)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77+/-3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (-)-isoproterenol induced stimulation of wild type receptor and addition of (-)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of beta-arrestin1 in mutant Asp104Lys promoted (39.75+/-2.19%) and knockdown of beta-arrestin1 by siRNA decreased (3.55+/-1.75%) internalization compared to Asp104Lys mutant of beta(1)-ARs. SIGNIFICANCE The present studies suggest that Asp104Lys mutant beta(1)-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and beta-arrestin1 play an important role for beta(1)-AR internalization.