Functional proteomics of G-protein-coupled receptors: Analysis of large-scale signaling devices

Signaling via G-protein-coupled receptors (GPCR) involves critical roles of gene expression, post-translational modifications (especially protein phosphorylation), and the formation/disassembly of macromolecular complexes that include GPCRs, protein kinases, phosphoprotein phosphatases, adapter molecules, and scaffolds. Dynamic analysis of GPCR signaling requires insight into the nature, stoichiometry, and assembly of signaling elements into complexes that provide for receptor activation, desensitization, down-regulation, re-sensitization, and translocation. The early work on molecular cloning of the GPCR signaling complexes was facilitated by industrial-scale purification and protein sequencing, most of the players are now known. The challenge to understanding the richness of GPCR signaling is largely confined to the "meso"-scale of analysis, with sizes of complexes well above the atomic, while not readily addressable by confocal microscopy, EM, and/or FRET analysis. Mass spectrometry has evolved into a powerful tool for modern strategies to the analysis of macromolecular signaling complexes. Enabling detailed analysis and positive identification of individual molecules by MALDI mass spectrometry using quadrupole capabilities for MALDI and tandem sequencing of fragments of unknown proteins enables inventory of all the elements within a signaling complex. In combination with structural biology, mass spectrometry will provide our greatest insight into how GPCRs propagate signals dynamically in vivo at the mesoscale.