Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

A new mild experimental approach for isolation of peptide membrane receptors and subsequent analysis of post-translational modifications is described. Endothelin receptors A and B were isolated on oligo(dT)-cellulose usingN-(ε-maleimidocaproyloxy)succinimide endothelin coupled to a protected (dA)-30-mer. This allowed a one-step isolation of the receptor from oligo(dT)-cellulose via variation solely of salt concentration. The identity of the receptor was confirmed by direct amino acid sequencing of electroblotted samples or by using antibodies against ETA and ETB receptors. The method used here is very fast, requires only very mild elution conditions and, for the first time, gave both ETA and ETB receptors concurrently in very good yield. Following enzymatic in-gel digestion, MALDI, and electrospray ion trap mass spectrometric analysis of the isolated endothelin B receptor showed phosphorylation at Ser-304, -418, -438, -439, -440, and -441. Further phosphorylation at either Ser-434 or -435 was observed. The endothelin B receptor is also palmitoylated at Cys residues 402 and 404. Phosphorylation of Ser304 may play a role in Hirschsprung's disease.