G-protein coupled receptor signaling (GPCR) is essential for regulating a large variety of hormonal, sensory and neuronal processes in eukaryotic cells. Because the regulation of these physiological responses is critical, GPCR signaling pathways are carefully controlled at different levels within the cascade. Phosducin-like protein 1 (PhLP1) can bind the G protein βγ dimer and participate in GPCR signaling. Recent evidence has supported the concept that PhLP1 can serve as a co-chaperone of the eukaryotic cytosolic chaperonin complex CCT/TRiC to mediate G βγ assembly. Although a general mechanism of PhLP1-mediated G βγ assembly has been postulated, many of the details about this process are still missing. Structural analysis of key complexes that are important intermediates in the G βγ assembly process can generate snapshots that provide molecular details of the mechanism beyond current understanding. We have isolated two important intermediates in the assembly process, the Gβ1-CCT and PhLP1-Gβ1-CCT complexes assembled in vivo in insect cells, and have determined their structures by cryo-electron microscopy (cryo-EM). Structural analysis reveals that Gβ1, representing the WD40 repeat proteins which are a major class of CCT substrates, interacts specifically with the apical domain of CCTβ. Gβ1 binding experiments with several chimeric CCT subunits confirm a strong interaction of Gβ1 with CCTβ and map Gβ1 binding to α-Helix 9 and the loop between β-strands 6 and 7. These regions are part of a hydrophobic surface of the CCTβ apical domain facing the chaperonin cavity. Docking the Gβ molecule into the two 3D reconstructions (Gβ1-CCT and PhLP1-Gβ1-CCT) reveals that upon PhLP1 binding to Gβ1-CCT, the quasi-folded Gβ molecule is constricted to a more native state and shifted to an angle that can lead to the release of folded Gβ1 from CCT. Moreover, mutagenesis of the CCTβ subunit suggests that PhLP1 can interact with the tip of the apical domain of CCTβ subunit at residue S260, which is a downstream phosphorylation target site of RSK and S6K kinases from the Ras-MAPK and mTOR pathways. These results reveal a novel mechanism of PhLP1-mediated Gβ folding and its release from CCT. The next important step in testing the PhLP1-mediated Gβγ assembly hypothesis is to investigate the function of PhLP1 in vivo. We have prepared a rod-specific PhLP1 conditional knockout mouse in which the physiological consequences of the loss of PhLP1 functions have been characterized. The loss of PhLP1 has led to profound consequences on the ability of these rods to detect light as a result of a significant reduction in the expression of transducin (Gt) subunits. Expression of other G protein subunits as well as Gβ5-RGS9-1 complexes was also greatly decreased, yet all of this occurs without resulting in rapid degeneration of the photoreceptor cells. These results show for the first time the essential nature of PhLP1 for Gβγ and Gβ5-RGS dimer assembly in vivo, confirming results from cell culture and structural studies.

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs that form various dimers with 12 Gγs and a 5th Gβ that dimerizes with the four regulator of G protein signaling (RGS) proteins of the R7 family. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1-4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results of Chapter 3 show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations, and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way. Chapter 4 discusses PhLP2, a recently discovered essential protein, and member of the Pdc family that does not play a role in G protein signaling. Several studies have indicated that PhLP2 acts as a co-chaperone with CCT in the folding of actin, tubulin, and several cell cycle and pro-apoptotic proteins. In a proteomics screen for PhLP2A interacting partners, α-tubulin, 14-3-3, elongation factor 1α, and ribosomal protein L3 were found. Further proteomics studies indicated that PhLP2A is a phosphoprotein that is phosphorylated by CK2 at threonines 47 and 52.