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

Abstract

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-(⑀-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 Bradykinin, a member of the kinin family (1), is a nonapeptide with diverse biological activities ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3). Bradykinin mediates its physiological effects by binding to and activation of the bradykinin B 2 receptor. Molecular cloning has revealed the primary structure of the B 2 receptor (4) and classified it as a member of the G protein-coupled receptor superfamily. The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is still rather little direct evidence at the protein level for attributes such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B 2 or other G protein-coupled receptors. Indeed phosphorylation sites for few G protein-coupled receptors have been mapped to date (10 -12), and most of these sites reflect the in vitro phosphorylation of the isolated receptor.We report here on the isolation of rat B 2 receptor from transfected Chinese hamster ovary (CHO) cells using oligo(dA) covalently linked to bradykinin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out under very mild conditions using oligo(dT) columns analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically separated receptor, subsequent peptide mass fingerprinting by matrix-assisted laser desorption...

Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin*♦

“…The latter effect may be due to hyperphosphorylation because further mutation of two phosphorylation sites restores basal phosphorylation levels and proper coupling of the receptor to G s (36). A steric regulatory role for palmitoylation is supported by reports that palmitoylation of the rat bradykinin ␤ 2 receptor at Cys 356 and phosphorylation of Tyr 352 are mutually exclusive, suggesting that phosphorylation can only occur upon depalmitoylation (38). In a heterologous system, light-dependent phosphorylation of rhodopsin lacking palmitate at position 323 was slightly lower than for wild-type rhodopsin, while rhodopsin lacking both palmitates behaved normally (28).…”

Mass Spectrometric Analysis of Agonist Effects on Posttranslational Modifications of the β-2 Adrenoceptor in Mammalian Cells

“…While this idea has been suggested previously for the B2AR (45), and shown for the R subunit of G s heterotrimeric G protein by direct determination of net palmitate incorporation (47), to our knowledge a direct analysis of net B2AR palmitoylation has not been accomplished previously in intact cells. We note that palmitoylation of another ligand-activated GPCR, the B2 bradykinin receptor, was shown previously by MS analysis from intact cells (6). In this study it was not possible to examine ligand effects on receptor PTMs because receptors were purified by affinity chromatography using an immobilized agonist peptide.…”

“…Important regulatory roles of PTMs have been amply demonstrated in studies of the light-activated GPCR rhodopsin as well as for a number of ligand-activated GPCRs (1)(2)(3)(4)(5)(6). PTMs of ligandactivated GPCRs are of particular interest because there is evidence, from studies of a number of GPCRs, that distinct agonists can mediate different effects on receptor signaling or regulation (7)(8)(9)(10)(11)(12)(13)(14).…”

“…Therefore, many studies carried out in intact cells have relied primarily upon site-directed mutagenesis combined with metabolic labeling to infer potential sites of PTMs and to examine agonist effects on the formation and/or turnover of PTMs (4,(19)(20)(21)(22). Indeed, with the exception of rhodopsin, relatively few studies have utilized direct readout methods to define GPCR PTMs occurring in cells or tissues (6,17,23,24). Furthermore, to our knowledge no previous studies have applied such methods to compare the effects of distinct agonists on net PTM of GPCRs in intact cells.…”

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…Functional proteomics of signal transduction pathways on growth-and bradikinin-receptors were investigated [126,127].…”