Functional proteomics of signal transduction by membrane receptors

Functional proteomic methods have been developed and applied to the investi gation of signal transduction systems involving platelet-derived growth fac tor (PDGF), endothelin and bradykinin receptors. Mouse fibroblast cells hav e been stimulated with PDGF or endothelin. Phosphorylation/dephosphorylatio n of several hundred proteins has been followed as a function of time follo wing stimulation using 2-D gel electrophoresis and anti-phosphotyrosine or anti-phosphoserine antibodies. Up to 100 of these proteins showed strong ch anges in phosphorylation with minutes of receptor stimulation. Identificati on of some of these proteins by mass fingerprinting using matrix-assisted l aser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and by partial peptide sequencing with ion trap electrospray mass spectrometry has identified proteins which were previously known to be associated with PDGF signaling, proteins which have been shown to be involved in other sign aling pathways, but not PDGF and proteins not previously associated with si gnal transduction. Parallel to these studies, new methods for rapid, single -step isolation of peptide receptors using a peptide coupled to a (dA)(30) oligonucleotide have been developed and applied to mass spectrometric studi es of post-translational modifications of the endothelin B and bradykinin B -2 receptors under in vivo conditions. Both receptors have been shown to un dergo extensive phosphorylation as well as palmitoylation. The patterns of post-translational modifications are more complex than previously recognize d and provide new indications of possible roles for these modifications in the regulation and response of these receptors.