Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B-2 receptor in Chinese hamster ovary cells

Rat bradykinin B-2 receptor from unstimulated Chinese hamster ovary cells t ransfected with the corresponding cDNA has been isolated, and subsequent ma ss spectrometric analysis of multiple phosphorylated species and of the pal mitoylation attachment site is described. Bradykinin B-2 receptor was isola ted on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy) succinimid e-Met-lys-bradykinin coupled to a protected (dA)(30)-mer. This allowed a on e-step isolation of the receptor on an oligo(dT)-cellulose column via varia tion solely of salt concentration. After enzymatic in-gel digestion, matrix -assisted laser desorption ionization and electrospray ion trap mass spectr ometric analysis of the isolated rat bradykinin B-2 receptor showed phospho rylation at Ser(365), Ser(371), Ser(378), Ser(380), and Thr(374). Further p hosphorylation at Tyr(362) and Tyr(161) was observed. Rat bradykinin recept or B-2 receptor is also palmitoylated at Cys(356). All of the phosphorylati on sites except for Tyr(161) cluster at the carboxyl-terminal domain of the receptor located on the cytoplasmic face of the cell membrane. Surprisingl y, many of the post-translational modifications were shown by MSn mass spec troscopic 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 palmitoylatio n at Cys(356). The last correlation may be involved in a receptor internali zation motif. Pairwise correlations and mutual exclusion of phosphorylation and palmitoylation suggest critical roles of multiple post-translational m odifications for the regulation of activity, coupling to intracellular sign aling pathways, and/or sequestration of the bradykinin receptor.