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Mechanisms of Ubiquitylation and Endoplasmic Reticulum- Associated Degradation of P23H Mutant Rhodopsin in Retinal Degeneration

  • Author(s): Chen, Allen PF
  • et al.
Abstract

Rhodopsin protein is the archetypal G-protein coupled receptor that is specifically and massively expressed in rod photoreceptor cells. Over 100 rhodopsin mutations lead to inheritable retinal disease. The P23H point mutation in rhodopsin leads to autosomal dominant retinitis pigmentosa, in which gradual death of rod photoreceptor cells results in night blindness and eventual total blindness. Both in vitro and in vivo systems demonstrate that P23H rhodopsin is misfolded and subjected to depletion by endoplasmic- reticulum-associated degradation (ERAD). Proper expression of mature rhodopsin is important not only for rod functionality, but also for its viability. Appropriately, we have previously shown that RhoP23H/P23H mice demonstrate an early degradation of rhodopsin that precedes rod photoreceptor death. However, the post- translational modifications that target rhodopsin for degradation are poorly understood. In the classical ERAD pathway, lysine residues are conjugated to poly-ubiquitin signals (ubiquitylation) for degradation by the proteasome. Here, we mutated every lysine residue (11 total) of P23H rhodopsin to arginine to test which ones are important for ubiquitylation. Using transient transfection of these rhodopsin constructs in the HEK293 human cell line, we found that a P23H rhodopsin with all lysine residues mutated (K-null P23H) is much less ubiquitylated than P23H rhodopsin with lysine residues intact. Cycloheximide blocking experiments demonstrate that the K-null P23H rhodopsin is more stable than P23H rhodopsin. Interestingly, rhodopsin constructs with singleton lysine mutations did not result in a significant change in ubiquitylation. In conclusion, ubiquitylation of multiple lysine sites on P23H rhodopsin may be involved in its ERAD processing

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