Mutations at position 125 in transmembrane helix III of rhodopsin affect the structure and signalling of the receptor

Anna Andrés, Ana Kosoy, Pere Garriga, Joan Manyosa

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25 Citations (Scopus)

Abstract

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with the retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mutations at this position were found to cause partial misfolding of the mutant receptors. We report here on a series of mutations at position 125 to further investigate the role of Leu125 in the correct folding and function of rhodopsin. In particular, the effect of the size of the substituted amino-acid side chain in the functionality of the receptor, measured as the ability of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N, L125I, L125H, L125P, L125T, L125D, L125E, L125Y and L125W. Most of the mutant proteins, expressed in COS-1 cells, showed reduced 11-cis-retinal binding, red-shifts in the wavelength of the visible absorbance maximum, and increased reactivity towards hydroxylamine in the dark. Thermal stability in the dark was reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantly reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substituted amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the β-ionone ring of 11-cis-retinal, is proposed to be an important residue in maintaining the correct structure of the chromophore binding pocket. Thus, bulky substitutions at this position may affect the structure and signalling of the receptor by altering the optimal conformation of the retinal binding pocket, rather than by direct interaction with the chromophore, as seen from the recent crystallographic structure of rhodopsin.
Original languageEnglish
Pages (from-to)5696-5704
JournalEuropean Journal of Biochemistry
Volume268
DOIs
Publication statusPublished - 3 Dec 2001

Keywords

  • G-protein-coupled receptor
  • Point mutations
  • Retinitis pigmentosa
  • Rhodopsin
  • Signal transduction

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