Abstract

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.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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