Search

The rapid phosphorylation and dephosphorylation of a variety of proteins downstream of the endothelin receptors A and B was investigated following stimulation of human lung fibroblasts with endothelin-1. Changes in the phosphorylation of proteins involved in the cell cycle, cytoskeleton, membrane channels, transcription, angiogenesis, and metabolism were observed. From observed changes in protein phosphatase 2A, CDC25 A, and caspase-2 precursor, a model for the promotion of cell cycle progression by ET-1 stimulation is proposed. This may offer insights into the mechanisms by which ET-1 exerts its mitogenic effects. The identities of the other proteins phosphorylated within 2 min of stimulation indicate that endothelin-1 also rapidly engages a diverse variety of other cellular responses.

Proteomic methods have been used to monitor changes in protein synthesis in the first 4 h following stimulation of human lung fibroblasts with endothelin-1. Using pulsed [(35)S]methionine labeling, about 70 proteins with altered protein synthesis could be detected, and the 35 proteins showing the largest changes were identified by mass spectrometry. The observed proteins included unexpected proteins such as Sox5, two isoforms of Rab14, Rab3A, translationally controlled tumor protein, and one protein of previously unknown function. There was a wide range of different kinetic behavior, and groups of functionally linked proteins such as Rab14, nucleophosmin,and cyclin-dependent kinase inhibitor 1B could be detected from similar kinetics. We propose that the functional proteomic methods are competitive with and have some advantages compared to expression profiling methods for monitoring gene expression.

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 using N-(epsilon-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.

Bovine lung endothelin-B receptor has been isolated in good yield with a new procedure involving the use of endothelin-1 coupled to iminobiotin with a long spacer and avidin-agarose affinity chromatography. Contrary to previous reports, evidence has been obtained that the native form of this receptor corresponds to the full-length transcript expected on the basis of cDNA clones. The binding of endothelin to a variety of shortened fragments of the full receptor suggests that the long N-terminal sequence of this receptor has very little influence on the binding of endothelin and that the main determinants of the endothelin binding site might be constituted by residues in the sixth, and possibly the seventh, transmembrane helices.