c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization

J Biol Chem. 2001 Apr 20;276(16):13240-7. doi: 10.1074/jbc.M011578200. Epub 2001 Jan 22.

Abstract

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.

MeSH terms

  • Adrenergic beta-Agonists / pharmacology*
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • CHO Cells
  • CSK Tyrosine-Protein Kinase
  • Carcinoma, Squamous Cell
  • Cricetinae
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Humans
  • Iodocyanopindolol / pharmacology
  • Isoproterenol / pharmacology
  • Luminescent Proteins / analysis
  • Luminescent Proteins / biosynthesis
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides, Antisense / pharmacology
  • Phosphorylation
  • Phosphotyrosine
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Receptors, Adrenergic, beta-2 / chemistry
  • Receptors, Adrenergic, beta-2 / drug effects
  • Receptors, Adrenergic, beta-2 / physiology*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / metabolism
  • Transfection
  • Tumor Cells, Cultured
  • beta-Adrenergic Receptor Kinases
  • src Homology Domains
  • src-Family Kinases

Substances

  • Adrenergic beta-Agonists
  • Luminescent Proteins
  • Oligodeoxyribonucleotides, Antisense
  • Receptors, Adrenergic, beta-2
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Phosphotyrosine
  • Iodocyanopindolol
  • Protein-Tyrosine Kinases
  • CSK Tyrosine-Protein Kinase
  • src-Family Kinases
  • CSK protein, human
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • Isoproterenol