Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well ...understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.
Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close ...locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.
The present work reports on a structural analysis carried out through different computer simulations of a set of rhodopsin mutants with differential functional features in regard to the wild type. ...Most of these mutants, whose experimental features had previously been reported Ramon et al. J Biol Chem 282, 14272-14282 (2007), were designed to perturb a network of electrostatic interactions located at the cytoplasmic sides of transmembrane helices 3 and 6. Geometric and energetic features derived from the detailed analysis of a series of molecular dynamics simulations of the different rhodopsin mutants, involving positions 134(3.49), 247(6.30), and 251(6.34), suggest that the protein structure is sensitive to these mutations through the local changes induced that extend further to the secondary structure of neighboring helices and, ultimately, to the packing of the helical bundle. Overall, the results obtained highlight the complexity of the analyzed network of electrostatic interactions where the effect of each mutation on protein structure can produce rather specific features.
Fourier transform infrared (FTIR) spectroscopy has been used for the detailed characterization and quantification of the secondary structure of bovine rhodopsin in native disc membranes. FTIR spectra ...were obtained in aqueous media, both in 1H2O and in 2H2O. Analysis of spectra by means of Fourier self-deconvolution, complemented with maximum likelihood restoration and Fourier derivative, has allowed the characterization of major amide I secondary structure-sensitive component bands of structural relevance which had not been detected before. In consequence, we show a richer secondary structure for rhodopsin than previously described. Our results indicate a total regular helix content around 51%, which would include not only the main alpha 1-type helix but also 3(10)-like helix. The presence of distorted helicoid sequences might furthermore increase to a certain extent the total helix amount. It is also indicated that a significant proportion of the amino acid residues are involved in extended/beta-structures and in reverse turns, as well as in "random" segments, which had not been directly demonstrated before. 61 +/- 4% of rhodopsin is determined to be solvent-accessible, which is a substantially higher value than previously reported. Helices account for most of the inaccessible moiety.
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.
A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins ...expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photo-intermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110--Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain.
Several motifs found in the third intracellular loop of the M(3) muscarinic receptor are critical for G protein activation and scaffold protein interaction. However, how multiprotein complexes form ...is not fully understood. A minigene encoding the third intracellular loop of the M(3) muscarinic receptor was constructed to explore whether peptides from this intracellular region could act as inhibitors of the muscarinic multiprotein complex formation and signaling. We found that this construct, when co-expressed with the M(3) receptor, has the ability to act as a competitive antagonist of G protein receptors and receptor-scaffold/accessory proteins. Transient transfection of human embryonic kidney-293 cells with DNA encoding the human M(3) and M(5) receptor subtypes results in a carbachol-dependent increase of inositol phosphate. Co-expression of the M(3) third cytoplasmic loop minigene dramatically reduces both carbachol-mediated G protein activation and inositol phosphate accumulation. Minigene expression also abrogates activation of M(3) and M(5) receptor mitogen-activated protein kinases pathway. Furthermore, minigene expression led to reduced AKT activation. These data, together with results of co-immunoprecipitation of different scaffold and kinase proteins, provide experimental evidence for the role for the third cytoplasmic loop of the human M(3) muscarinic receptor in G-protein activation and multiprotein complex formation.
G‐protein‐coupled receptors are integral membrane proteins which constitute the largest family of signal transduction molecules participating in the majority of normal physiological processes. ...G‐protein‐coupled receptors are responsible for the control of enzyme activity, ion channels and vesicle transport, and they respond to a wide variety of stimuli, like signals involved in sensory systems such as vision, taste and olfaction, but also to a diverse set of chemical signals such as lipids, hormones, neurotransmitters, amino acids, nucleotides, peptides and proteins. This family of receptors is being widely studied because of its potential use as pharmacological targets in drug development, and recently also for its potential use in the development of novel biosensors. G‐protein‐coupled receptors are specifically designed to fold and function in a lipid bilayer environment, where these membrane proteins are remarkably stable and achieve their optimal performance. The currently used technology for the purification of G‐protein‐coupled receptors consists in their extraction from the cell membrane and solubilization into detergent micelles. A common drawback of this strategy is that G‐protein‐coupled receptors solubilized in typical detergents show rather poor conformational stability, which may result in relatively rapid inactivation. The poor stability of detergent‐solubilized samples renders many membrane proteins biochemically intractable. This precludes the determination of a high‐resolution structure and imposes severe limitations for the development of applications. Thus, the enhancement of the stability of G‐protein‐coupled receptors is a major issue in order to facilitate structural determination and to unravel their potential in biotechnological applications. This work provides a brief overview of some current advances in the experimental methods for stabilizing G‐protein‐coupled receptors that can also be extended to other types of membrane proteins.
Inorganic salts and novel solubilizing systems enhance the structural stability of membrane proteins opening new avenues for biotechnological applications. Stabilized G‐protein‐coupled receptors can be used for the development of novel biosensors and in biomedical research for clinical diagnostics.
No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca super(2) super(+)/recoverin upon ...phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca super(2) super(+)/recoverin than wild-type rhodopsin. This suggests an involvement of Ca super(2) super(+)/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca super(2) super(+)/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.
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