Mecanisme d'activació de la Rodopsina i reconeixement molecular de la proteïna Gt

Abstract

Rhodopsin (Rho) is the visual photoreceptor responsible for dim light vision. This receptor, that is located in the rod cell of the retina, has seven transmembrane helices and is a prototypical member of the G protein coupled receptors (GPCR) superfamily, being the only member of this superfamily whose structure has been resolved by X-ray chrystallography. The study of GPCR is of outstanding pharmacological interest because they are involved in a wide variety of physiological and pathophysiological processes. Structural and functional studies of Rho should provide clues about common structural motifs in GPCR and allow us to elucidate the molecular bases of a proposed common activation mechanism. Besides, the study of Rho mutants associated with retinal diseases, such as retinitis pigmentosa (RP) provides information about the molecular mechanism of this pathology. The main goal of this work is unraveling the structural details underlying the molecular mechanism of Rho activation and its interaction with transducin. To this aim we have analyzed the structural and functional role of helices I and II of Rho, as well as the role of retinal in the photoactivation process of the receptor. We have also tried to determine the amino acids involved in the specificity of G protein recognition. We have studied the adRP mutants G51A, G51V and G89D, as well as non-natural mutations at these positions, in order to determine the role of helices I and II in Rho structure and conformational changes upon light activation. The detailed characterization of mutations at 51 position shows that this position is very sensitive to the size of the introduced side chain. The increase volume of side chain of the introduced amino acid can be correlated with dark and active conformation (MetaII) stability and eventually with the capacity to activate the G-protein transducin. The study of mutations at 89 position suggests that the charge effect is more important for mutations in this position. Additionally, G51V and G89D mutants showed an abnormal photobleaching, with formation of an altered photointermediate which is in equilibrium to MetaII. We suggest that G51V and G89D mutants alter the proposed equilibrium between MetaIa, MetaIb and MetaII to favour the MetaIb photointermediate, and this fact together with the observed MetaII instability could be the molecular defects that caused RP. Wt Rho was regenerated with 11-cis-7 methylretinal (7-methyl-Rho), to gain insights into the opsin-retinal coupling process and to determine the role of retinal in the photoactivation process. 7-methyl-Rho showed chromophore formation similar to Rho but an abnormal photobleaching behaviour with formation of an altered photointermediate which is in equilibrium to MetaII photointermediate. The methyl group at C7 of retinal would introduce a structural change at the vicinity of Met-207 that would result in receptor trapping at the MetaIa conformation during the photoactivation process. MetaIa, MetaIb and MetaII equilibrium would be controlled by a network of complex interactions between helices I, II and VII of rhodopsin and also by opsin and retinal interactions. In another approach, we constructed and expressed Rho-m3 mutants at intracellular loops C-II and C-III. These mutants showed chromophore formation and photobleaching behaviour similar to Wt Rho. Single point mutants at the C-II loop showed transducin activation like Wt Rho with the exception of V138S mutation that caused a reduction in transducin activation like mutants at C-III loop and conjugated mutants at C-II and C-III loops. None of these mutants showed Giaq activation with regard to Wt protein. The results suggest that Val-138, Val-227, Val-250, Val-254 and Ile-255 amino acids of Rho participate in transducin binding and/or activation, and that hydrophobic interactions involving these residues would be an important factor mediating Rho-transducin interaction.