title: Mutagenesis of important amino acid reveals unconventional homologous internalization of beta(1)-adrenergic receptor.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
authors: Murad
authors: M
authors: Hossain
authors: Mamunur
authors: M
authors: Rashid
authors: Mohiuddin Ahmed
authors: MA
authors: Bhuiyan
authors: Takashi
authors: T
authors: Nakamura
authors: Masanobu
authors: M
authors: Ozaki
authors: Takafumi
authors: T
authors: Nagatomo
mesh_terms: D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics*; D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011433:Propranolol / Q000378:metabolism; D011869:Radioligand Assay; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D008024:Ligands; D018342:Receptors, Adrenergic, beta-1; D001224:Aspartic Acid; D011433:Propranolol; D000242:Cyclic AMP; D008239:Lysine; D007545:Isoproterenol