Phosducin-like protein (PhLP), a G protein beta gamma subunit dimer binder and G protein signaling regulator, was suggested to regulate the activity of cytosolic chaperonin CCT by their high affinity interaction. In the present study, the three-dimensional structure of PhLP:CCT complex has been solved by cryoelectron microscopy. PhLP was found to bind only one of the chaperonin rings with both N- and C-terminal domains. It spans the central folding cavity of CCT and interacts with two opposite sides of the top apical region, inducing the constraining of the entry of the folding cavity. These findings support a putative role of PhLP as a co-chaperone similar to prefoldin. Docking studies with the atomic model of PhLP generated from several known structures of the homologous phosducin (Pdc) together with the immuno-EM studies have provided more details of the complex structure and predicted some regions of PhLP and the subunits of CCT involved in the interaction. Taking advantage of the fact that Pdc is highly homologous to PhLP but lack of binding to CCT, the regions of PhLP involved in the interaction with CCT were determined by testing various PhLP/Pdc chimeric proteins in the CCT binding assay. In the other part of this dissertation, the physiological role of PhLP in G protein signaling was investigated. Cellular expression of PhLP was blocked using RNA interference targeting PhLP. Together with overexpression of PhLP variants and kinetic studies of G protein beta gamma dimer formation, PhLP was determined to be a positive mediator of G protein signaling and essential for G protein beta gamma dimer expression and dimer formation. Phosphorylation of PhLP at serines 18—20 by protein kinase CK2 was required for G protein beta gamma dimer formation, while a high-affinity interaction of PhLP with CCT appeared unnecessary. Interestingly, G protein beta subunit was found to interact with CCT by co-immunoprecipitation and PhLP over-expression increased the binding of G protein beta subunit to CCT. These results suggest that PhLP and CCT act as co-chaperones in the folding and assembly of the G protein beta gamma subunit dimer by forming a ternary PhLP-Gbeta-CCT complex that is a necessary intermediate in the assembly process.

Phosducin-like protein (PhLP) binds G-protein beta gamma subunits and is thought to assist in assembly of the G-protein beta gamma dimer. Phosphorylation of PhLP at serine residues 18-20 by the casein kinase 2 (CK2) appears to play an essential role in this process. PhLP has also been shown to interact with the chaperonin containing TCP-1 (CCT) atop its apical domain, not entering the substrate folding cavity. However, the physiological role of the PhLP-CCT interaction in G-protein beta gamma dimer formation remains unclear. This study addresses the mechanism of G-protein beta gamma assembly by exploring the specific roles of CCT and CK2 phosphorylation of PhLP in the assembly process. Both overexpressed and endogenous Gbeta were shown to co-immunoprecipitate with CCT to a similar extent as PhLP, indicating that CCT may be involved in the folding of Gbeta. In addition, Ggamma overexpression enhanced the binding of PhLP to CCT, suggesting the formation of a ternary PhLP-Gbeta-CCT complex. In contrast, overexpression of PhLP caused the release of G-beta from CCT. This release was blocked by a PhLP S18-20A variant that lacks the S18-20 CK2 phosphorylation site. PhLP S18-20A has been previously shown to negatively affect the G-protein beta gamma dimer formation, suggesting a correlation between PhLP-mediated release of Gbeta from CCT and G protein beta gamma assembly. Experiments investigating the role of Ggamma in this process show that Ggamma does not interact with CCT nor is it the essential factor in the release of Gbeta from CCT. A new model is therefore proposed for the G-protein beta gamma subunits' assembly involving the formation of a PhLP-Gbeta-CCT ternary complex followed by the release of a phosphorylated PhLP-Gbeta complex from CCT. In the PhLP-Gbeta complex, the Ggamma binding face of Gbeta is exposed, allowing for the formation of the G-protein beta gamma dimer.