Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

Rat bradykinin B 2 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 B 2 receptor was isolated on oligo(dT)-cellulose using N -(ε-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 B 2 receptor showed phosphorylation at Ser 365 , Ser 371 , Ser 378 , Ser 380 , and Thr 374 . Further phosphorylation at Tyr 352 and Tyr 161 was observed. Rat bradykinin receptor B 2 receptor is also palmitoylated at Cys 356 . All of the phosphorylation sites except for Tyr 161 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 MS n mass spectroscopic analysis to be correlated pairwise, e.g. diphosphorylation at Ser 365 and Ser 371 , at Ser 378 and Ser 380 , and at Thr 374 and Ser 380 as well as mutually exclusive phosphorylation at Tyr 352 and palmitoylation at Cys 356 . 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.