Biochem Blogs

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Role of Phosducin Like Protein 1 in G protein assembly in retinal rod photoreceptors

Graduate student Dipti Paudel

Lai, Chun Wan J., et al., Phosducin-like protein 1 is essential for G protein assembly and signaling in retinal rod photoreceptors. The Journal of neuroscience. May 1, 2013. 33(18):7941-7951.

Rod and cone cells are photoreceptors present in the retina of the eye. Cone cells primarily function in photopic (well-lit condition) vision whereas rods function in scotopic (intermediate light conditions) vision. Rods are more sensitive than cones, requiring only one photon for activation of intracellular signaling. G protein is a heterotrimeric protein associated with seven transmembrane G protein coupled receptors (GPCRs) and is signaling protein in rod and cone cells. When a photon binds to the GPCR, exchange of GDP to GTP activates Gα subunit separating it from the Gβy obligate dimer. The dimer is then able to perform downstream signaling through second messenger system. The original assembly of the Gβy dimer requires at least two co-chaperones: Chaperonin-Containing T complex polypeptide 1 (CCT1) and Phosducin Like Protein 1 (PhLP1). To test the role of PhLP1 in dimer formation, in vivo knockouts of PhLP1 from mice retinal rod photoreceptors were introduced and monitored for G protein expression.

First, knockout mice were created using Lox-Cre recombinase system. After two generations, mice with deleted PhLP1 (homozygous knockouts) were formed along with heterozygous and wild type mice.  Using one month old mice, immunohistochemistry was used to confirm the deletion of PhLP1 in rod cell outer segments. Using immunoblot of retinal extracts cre mediated knockouts were determined to have lost 80% of PhLP1 gene expression after 35 days. Second, the researchers looked at the expression of various G protein subunits in the absence of PhLP1. Using immunohistochemistry on retinal tissue, a disappearance of Gαt1, 1, Gγ1, and RGS9 was observed. Since Gβ4 and Gb5 are usually present in the inner nuclear layer and not the outer rod segments, their expression remained unchanged. Rhodopsin levels were also unchanged, indicating that only the G proteins were affected. To further confirm the expressions, extracts from whole retinal and retinal outer segments were immunoblotted for G proteins as well as varied cone and rod proteins. The levels of most cone and rod proteins remained unchanged but Phosducin (Pdc), a protein used for stabilization of the Gβγ complex, contained decreased levels in PhLP1 knockouts. A co-immunoprecipitation study of Pdc indicated no dimer formation in the homozygous knockout. To test the visual acuity and sensitivity of mice in the different study groups, the optomeric head turning response of wildtype and homozygous knockout mice to scotopic and photopic conditions were measured. The results indicated low response for homozygous knockout mice in scotopic conditions and an unchanged response in photopic conditions further confirming that only rod cells were affected.

In conclusion, this study showed that deletion of the co-chaperon PhLP1 decreased the expression of G proteins in the outer rod segment of the retina and inhibited assembly of the Gβγ complex.

Dipti_2

Figure 1: The proposed mechanism for G protein heterotrimer assembly and the effects of PhLP1deletion on the assembly (shown in red).