Abstract
The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.
Highlights
The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa
G51V had an absorbance maximum slightly red-shifted to 502 nm, whereas G51L had it blue-shifted to 497 nm (Table II)
Mutations at Position 51 in Helix I—Gly-51 in the transmembrane helix I of rhodopsin is part of a GXXXN motif conserved in the G-protein-coupled receptors (GPCR) superfamily
Summary
A NOVEL INTERPLAY EVIDENCED BY MUTATIONS AT GLY-51 AND GLY-89 IN THE TRANSMEMBRANE DOMAIN*. We started a detailed characterization of ADRP mutants in the transmembrane region of rhodopsin with a 2-fold interest, (i) elucidation of the molecular mechanism of the disease and (ii) structural and functional information, from the study of these mutations that might be relevant to other members of the GPCR superfamily. These studies showed that G51V forms chromophore like wild-type rhodopsin (WT), whereas G89D could only form it partially [22] We took these previous studies as a starting point for a detailed characterization of the helical environment of Gly-51 and Gly-89 in transmembrane helices I and II of rhodopsin by analyzing a series of mutants at these positions. Overall our results provide evidence for the presence of specific interhelical interactions, in the region delimited by helices I, II and VII in the transmembrane domain of rhodopsin, that are important for the stability and the function of the receptor
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