Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B2 receptor in Chinese hamster ovary cells

J Biol Chem. 1999 Mar 26;274(13):8539-45. doi: 10.1074/jbc.274.13.8539.

Abstract

Rat bradykinin B2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B2 receptor was isolated on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA)30-mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B2 receptor showed phosphorylation at Ser365, Ser371, Ser378, Ser380, and Thr374. Further phosphorylation at Tyr352 and Tyr161 was observed. Rat bradykinin receptor B2 receptor is also palmitoylated at Cys356. All of the phosphorylation sites except for Tyr161 cluster at the carboxyl-terminal domain of the receptor located on the cytoplasmic face of the cell membrane. Surprisingly, many of the post-translational modifications were shown by MSn mass spectroscopic analysis to be correlated pairwise, e.g. diphosphorylation at Ser365 and Ser371, at Ser378 and Ser380, and at Thr374 and Ser380 as well as mutually exclusive phosphorylation at Tyr352 and palmitoylation at Cys356. The last correlation may be involved in a receptor internalization motif. Pairwise correlations and mutual exclusion of phosphorylation and palmitoylation suggest critical roles of multiple post-translational modifications for the regulation of activity, coupling to intracelluar signaling pathways, and/or sequestration of the bradykinin receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cricetinae
  • Mass Spectrometry
  • Molecular Sequence Data
  • Palmitates / metabolism*
  • Peptide Fragments / chemistry
  • Phosphopeptides / analysis
  • Phosphorylation
  • Phosphoserine / analysis
  • Phosphothreonine / analysis
  • Phosphotyrosine / analysis
  • Protein Processing, Post-Translational
  • Rats
  • Receptor, Bradykinin B2
  • Receptors, Bradykinin / chemistry
  • Receptors, Bradykinin / metabolism*
  • Transfection
  • Trypsin / metabolism

Substances

  • Palmitates
  • Peptide Fragments
  • Phosphopeptides
  • Receptor, Bradykinin B2
  • Receptors, Bradykinin
  • Phosphothreonine
  • Phosphoserine
  • Phosphotyrosine
  • Trypsin