Abstract
Autosomal dominant retinitis pigmentosa (ADRP) mutants (T4K, N15S, T17M, V20G, P23A/H/L, and Q28H) in the N-terminal cap of rhodopsin misfold when expressed in mammalian cells. To gain insight into the causes of misfolding and to define the contributions of specific residues to receptor stability and function, we evaluated the responses of these mutants to 11-cis-retinal pharmacological chaperone rescue or disulfide bond-mediated repair. Pharmacological rescue restored folding in all mutants, but the purified mutant pigments in all cases were thermo-unstable and exhibited abnormal photobleaching, metarhodopsin II decay, and G protein activation. As a complementary approach, we superimposed this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) that forms a disulfide bond. This approach restored folding in T4K, N15S, V20G, P23A, and Q28H but not T17M, P23H, or P23L. ADRP mutant pigments obtained by disulfide bond repair exhibited enhanced stability, and some also displayed markedly improved photobleaching and signal transduction properties. Our major conclusion is that the N-terminal cap stabilizes opsin during biosynthesis and contributes to the dark-state stability of rhodopsin. Comparison of these two restorative approaches revealed that the correct position of the cap relative to the extracellular loops is also required for optimal photochemistry and efficient G protein activation.
Highlights
Retinitis pigmentosa mutations in the N terminus of rhodopsin lead to misfolding
Pharmacological Chaperone Rescue of Misfolded Autosomal dominant retinitis pigmentosa (ADRP) Rhodopsin Mutants—HEK-293S cells transfected with ADRP rhodopsin mutants were incubated in the presence or absence of Expression levels and spectral properties of mutant pigments
The N2C/D282C rhodopsin G protein transducin (GT) activation level was reduced by 10% in the presence of DTT, whereas the activation rates with the single N2C or D282C mutant treated with DTT were ϳ5% lower
Summary
Retinitis pigmentosa mutations in the N terminus of rhodopsin lead to misfolding. Results: Pigments obtained by pharmacological rescue remain defective, whereas some of those obtained by disulfide bondmediated repair regain function. To test our hypothesis directly, we introduced a disulfide bond in an attempt to tether the damaged cap to the 7TM domain This was achieved by superimposing this panel of ADRP mutants onto a rhodopsin background containing a juxtaposed cysteine pair (N2C/D282C) known to form a disulfide bond (Cys-2– Cys-282) that increases the thermostability of rod opsin [23]. For a subset of these ADRP mutants, the N2C/D282C background restored folding of rod opsin, and the resulting pigments had much improved thermostability, photobleaching behavior, and activation of the G protein transducin (GT) These findings highlight how the extracellular domain of rhodopsin has evolved, under selection pressure constraints requiring high sensitivity vision in dim light, to contribute to receptor stability and signaling efficiency
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