Heterotrimeric G proteins play a fundamental role in cell signaling by shuttling the ligand binding information received by G protein-coupled receptors (GPCRs) on the cell surface to effector enzymes and ion channels on the intracellular face of the plasma membrane. By virtue of an ability to bind the G protein beta/gamma subunit dimer, the phosducin (Pdc) family of proteins has been thought for some time to participate in G protein signaling. However, their role in this process has remained elusive. It has recently been reported that a member of the phosducin family, phosducin-like protein (PhLP1), is required for assembly of the G beta/gamma dimer from its nascent polypeptides. Other members of the Pdc family, PhLP2 and PhLP3, do not bind G beta/gamma, but they have also been implicated in other protein folding processes. Given these new findings, it appears that the primary function of PhLPs may be to assist in the folding of nascent polypeptides and the assembly of these proteins into complexes. The proposed experiments seek to determine the molecular mechanism by which PhLP1 catalyzes G beta/gamma assembly and to elucidate the role of PhLP2 and PhLP3 in protein folding. To this end, the specific aims of the proposal are to: 1) Define the molecular mechanism of phosphorylation-dependent release of PhLP1-Gbeta from CCT. 2) Identify conformational changes in the PhLP1-Gbeta-CCT complex induced by PhLP1 phosphorylation. 3) Determine the effects of PhLP1 on the assembly of different Gbeta/gamma subunit combinations. 4) Assess the effects of PhLP3 phosphorylation on actin and tubulin folding. 5) Determine the function of PhLP 2A in 14-3-3 protein and alpha-tubulin folding. The methods used to achieve these aims are multi-disciplinary and they include RNA interference, cell transfection, immunoprecipitation, site-directed mutagenesis, protein folding and subunit assembly assays, cryo-electron microscopy, X-ray crystallography and mass spectrometric identification of proteins and their phosphorylation sites. These proposed studies address fundamental issues in protein folding in general and G protein beta/gamma dimer formation in particular. As such, they will provide information important in understanding the pathologies associated with protein misfolding such as Alzheimer's, prion and Huntington's disease as well as in the development of novel therapeutic targets in G protein pathways.