Semester

Summer

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Maxim Sokolov

Abstract

Phosducin (Pdc) and phosducin-like protein 1 (PhLP1) are homologous proteins of the phosducin gene family that specifically interact with heterotrimeric G proteins. Both Pdc and PhLP1 are expressed in photoreceptors, sensory neurons of the retina responsible for acquisition of visual information; however their functions in these cells remains enigmatic. Photoreceptors maintain remarkably high and tightly controlled levels of the heterotrimeric G protein, transducin, utilized by these cells for visual signal transduction. Our central hypothesis was that Pdc and PhLP1 are engaged in the maintenance of transducin homeostasis in the photoreceptors.;To test this hypothesis, we have studied phosphorylation of Pdc, which regulates its interaction with transducin in a light-dependent manner. For that, we have determined light-dependence, time-course, and localization of phosphorylated Pdc within the photoreceptor cell under various physiologically relevant conditions of illumination. We found that phosphorylation of Pdc in vivo occurs in a site- and compartment-specific manner, and is specifically enriched in the border between the inner and outer segments of rod photoreceptors. These findings are described in Part I of Chapter 2.;An abundance of Pdc phosphorylation at the entrance to the outer segment allowed us to hypothesize that Pdc regulates trafficking of transducin to this compartment. To test our hypothesis directly, we generated transgenic mice expressing Pdc lacking the principal phosphorylation site, serine 54 and serine 71, under the control of a rhodopsin promoter. Using this Pdc phosphorylation mutant, and transgenic mice expressing full-length Pdc as a control, we compared the rates of transducin trafficking to the rod outer segments, and found that phosphorylation of Pdc significantly accelerates trafficking of transducin to the rod outer segments. This ongoing research project is described in Part II of Chapter 2.;To explore the role of PhLP1, we have suppressed its endogenous activity in photoreceptors using transgenic overexpression of a dominant-negative N-terminally truncated splice-isoform of PhLP1. We found that suppressing PhLP1 activity triggered fast and severe photoreceptor degeneration, apparently due to a profound disruption in transducin expression. These findings, described in Chapter 3, strongly support our hypothesis that PhLP1 plays a central role in the post-translational stabilization of transducin subunits, which is essential for visual function and rod viability.;In summary, our findings have demonstrated that phosducin and phosducin-like protein 1 function as specific chaperones in the folding, assembly and trafficking of transducin subunits in photoreceptors.

Download

Share

COinS