Mutagenesis of Important Amino Acid Reveals Unconventional Homologous Internalization of Beta(1)-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 (beta ARK1) 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 [H-3] 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. (C) 2009 Elsevier Inc. All rights reserved.