Functional proteomics of signal transduction by membrane receptors.
Functional proteomic methods have been developed and applied to the investigation of signal transduction systems involving platelet-derived growth factor (PDGF), endothelin and bradykinin receptors. Mouse fibroblast cells have been stimulated with PDGF or endothelin. Phosphorylation/dephosphorylation of several hundred proteins has been followed as a function of time following stimulation using 2-D gel electrophoresis and anti-phosphotyrosine or anti-phosphoserine antibodies. Up to 100 of these proteins showed strong changes in phosphorylation with minutes of receptor stimulation. Identification of some of these proteins by mass fingerprinting using matrix-assisted laser 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 signaling pathways, but not PDGF and proteins not previously associated with signal 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 studies of post-translational modifications of the endothelin B and bradykinin B2 receptors under in vivo conditions. Both receptors have been shown to undergo extensive phosphorylation as well as palmitoylation. The patterns of post-translational modifications are more complex than previously recognized and provide new indications of possible roles for these modifications in the regulation and response of these receptors.
Godovac-Zimmermann
J
J
Center for Molecular Medicine, University College London, UK. j.godovac-zimmermann@ucl.ac.uk
Soskic
V
V
Poznanovic
S
S
Brianza
F
F
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D018002:Receptors, Bradykinin / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000378:metabolism*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016232:Endothelins; D010982:Platelet-Derived Growth Factor; D018002:Receptors, Bradykinin; D017466:Receptors, Endothelin