All ETDs from UAB

Advisory Committee Chair

Alecia K Gross-Gutierrez

Advisory Committee Members

Tim W Kraft

Vladimir Parpura

Elizabeth S Sztul

Bradley K Yoder

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Rhodopsin is a prototypical G-protein coupled receptor whose biological function has been well worked out, yet little is known about the mechanism by which it is so efficiently transported to the rod outer segment (ROS) after synthesis. If rhodopsin is not efficiently transported to the ROS, rod cells will apoptose leading to cone cell apoptosis as well which results in the blinding disease retinitis pigmentosa. Work has begun to elucidate proteins responsible for transporting rhodopsin to the ROS using Xenopus laevis as a model since the photoreceptors of this amphibian are large and transgenic animals are relatively easy to prepare. However, many of the key players have yet to be identified and the exact role of many of the known players is still poorly understood. Furthermore, the high concentrated of rhodopsin in the ROS compared to the low amounts of rhodopsin within the rod inner segment (RIS) overwhelms the detectability of rhodopsin in the RIS thereby limiting the characterization of rhodopsin movement toward the ROS when it localizes properly or toward the plasma membrane when it localizes improperly. Herein, we introduce photoactivatable GFP fused to the rhodopsin C-terminus (rho-paGFP) with the last eight amino acids of rhodopsin (1D4) appended the C-terminus of the fusion (rho-paGFP-1D4) for use in monitoring rhodopsin transport. We first compile routinely used methods of purifying rhodopsin and characterizing its function. We then demonstrate that rho-paGFP-1D4 folds and functions similarly to rhodopsin. We additionally show that rho-paGFP-1D4 localizes correctly to the cilium of cultured IMCD cells and to the ROS of rod cells. To probe rhodopsin transport within the rod inner segment, we generated a knock-in mouse expressing rho-paGFP-1D4. We monitored rho-paGFP-1D4 transport ex-vivo alone, expressed with rhodopsin, or expressed with the rhodopsin truncation mutant Q344ter rhodopsin. We then knocked down active transport with the addition of sodium azide (NaN3) to differentiate active and passive rhodopsin transport. We confirm that rho-paGFP-1D4 preferentially moves toward the ROS as opposed to the synapse in all three models, with a slight larger percentage moving toward the synapses in the rho-paGFP-1D4/Q344ter retinas. We further reveal that all rho-paGFP-1D4 transport was reduced upon addition of an active transport blocking agent, NaN3. While percent reduction in transport toward the ROS was model specific, reduction in transport toward the synapse was similar between all three models suggesting that rhodopsin transport toward the synapse is independent of rhodopsin's sorting motif.

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