The Third Intracellular Loop of truman ${\beta}_2$-adrenergic Receptor Expressed in E. coli Decreased Binding Affinity of Isoproterenol to ${\beta}_2$-adrenergic Receptor

  • Shin, Jin-Chul (Research Institute of Dong Shin Pharmaceutics) ;
  • Shin, Chan-Young (College of Pharmacy and Institute for Molecular Biology and Genetics, Seoul National University) ;
  • Lee, Mi-Ok (College of Pharmacy and Institute for Molecular Biology and Genetics, Seoul National University) ;
  • Lee, Sang-Bong (Lab. of Cell Signaling, National Institute of Health) ;
  • Ko, Kwang-Ho (College of Pharmacy and Institute for Molecular Biology and Genetics, Seoul National University)
  • Published : 1996.03.01

Abstract

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

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