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

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with t he retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mu tations at this position were found to cause partial misfolding of the muta nt receptors. We report here on a series of mutations at position 125 to fu rther 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 abili ty of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N. L125I, L 125H, L125P, L125T, L125D. L125E, L125Y and L125W. Most of the mutant prote ins, expressed in COSA cells, showed reduced 11-cis-retinal binding, red-sh ifts in the wavelength of the visible absorbance maximum. and increased rea ctivity towards hydroxylamine in the dark. Thermal stability in the dark wa s reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantl y reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substitut ed amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the beta -ionone r ing of 11-cis-retinal, is proposed to be an important residue in maintainin g the correct structure of the chromophore binding pocket. Thus, bulky subs titutions at this position may affect the structure and signallling 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.