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PubMed1998

Characterization of canine photoreceptor phosducin cDNA and identification of a sequence variant in dogs with photoreceptor dysplasia.

Photoreceptor dysplasia (pd) is an autosomal recessive disease of miniature schnauzer dogs causing retinal degeneration. The disease is a homologue of retinitis pigmentosa, a group of genetically heterogeneous diseases, causing blindness in humans. A subtraction library was prepared from retinas of pd affected and age-matched normal control dogs to isolate de novo candidate genes for further examination. From the subtraction library, cDNA for phosducin (PDC), a member of the phototransduction pathway, was isolated as a transcript expressed at a higher level in the affected retina. First, the normal canine PDC cDNA was characterized to evaluate the PDC gene in the pd-affected retina. The characterized region of normal PDC cDNA spans 1258 nucleotides (nt) that include 738 nt of coding sequence predicted to encode a protein (Mr=28 209) of 245 amino acids (aa). Over the coding region, PDC shares 86-95% nt sequence identity and 90-95% identity in the deduced aa sequence with homologous mammalian sequences. A major transcript (1.9 kb) was observed only in retina by Northern analysis, but low levels of transcript were detected in brain, liver and kidney by reverse transcription and polymerase chain reaction. Retinal immunocytochemistry showed that PDC was detected only in rod photoreceptors, mainly in the inner segment and perinuclear region. By Northern blot analysis, increased PDC expression was observed in pre-degenerate affected retina relative to the age-matched normal. In pd- affected miniature schnauzer pedigree, a missense mutation was detected in codon 82 (CGA to GGA) that would create a non-conservative substitution (Arg to Gly) in close vicinity to the residue (Glu 85) which directly interacts with the betagamma-subunits of transducin. Only pd-affected dogs were found to be homozygous for the mutant allele, and none among 48 dogs tested from 20 other dog breeds had this allele, suggesting that the mutation could be causally associated with pd in miniature schnauzers. However, since some affected dogs are heterozygous for the mutant allele, and some are homozygous for the wild-type allele, this putative PDC missense mutation, if it is indeed a disease causing mutation, does not account entirely for the genetics of inherited retinal degeneration in the miniature schnauzer breed.

Amino Acid Sequence↗
PubMed1999

Cloning of the human phospholipase A2 activating protein (hPLAP) gene on the chromosome 9p21 melanoma deleted region.

Cutaneous malignant melanoma (CMM) is a common skin cancer. About 50% of CMM sporadic tumours have lost one copy of the chromosome 9p21 region. To identify genes involved in the initiation and/or progression of CMM we have characterised the 9p21 melanoma deleted region and screened the human expressed sequence tag (EST) databases (dbEST) to search for expressed genes. We have identified the gene that encodes the human orthologue of the rat phospholipase A2 activating protein (PLAP). PLAP was considered a potential candidate to be involved in malignant melanoma because it maps to the critical region for CMM and because the PLA2 gene has been identified as a modifier of the APC gene, responsible for the adenomatous polyposis phenotype in the mouse. PLAP encodes a protein of 738 amino acids and has a high DNA (90%) and protein (97%) sequence similarity with the rat and mouse PLAP protein. PLAP has a region of WD40 repeats in the amino-terminus, which allows us to include this protein in the superfamily of beta-transducin proteins. Northern blot hybridisation gave a fragment of 4.5 kb, with higher expression in heart compared to other tissues. PLAP was localised at chromosome 9p21, between marker AFM218xg11 and TEK. SSCP analysis of the coding region of PLAP revealed no variants in the studied samples, but one of six CMM samples analysed by RT-PCR showed specific inactivation of PLAP. Despite PLAP's important role in mediating several cellular responses and its localisation to the chromosome 9p21 region deleted in CMM, it is unlikely that point mutations or deletions in the coding region of PLAP are responsible for the initiation or progression of CMM. Further studies on PLAP inactivation should be performed to clarify its potential involvement in CMM.

Amino Acid Sequence↗
PubMed1997

Effect of different illumination conditions and ionic environment on the guanylate cyclase activity in retina, optic nerve and optic chiasm of the rat.

Guanylate cyclase is sensitive to changes of light and dark periods in incubated extracts obtained from soluble fractions of the retina, optic nerve and optic chiasm. The changes in guanylate cyclase activity found, about 100-fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibits strongly the retinal guanylate cyclase activity, while it increases the activity of this enzyme in the optic nerve. A generalized photo-inhibited response of guanylate cyclase was observed in all studied tissues in animals adapted to the dark. This suggests that light could act as a double stimulus gating the central circuit which promotes the hydrolysis of cGMP via cGMP phosphodiesterase-rhodopsin-transducin cascade, and by direct inhibition of the retinal guanylate cyclase activity. Finally, different responses have been observed in the guanylate cyclase activity in relation with the ion exposure depending on the studied tissue. In summary, all indicate an important role for the soluble guanylate cyclase activity in retina, and other tissues involved in the visual process such as optic nerve and optic chiasm, which have not been examined until now.

Adaptation, Physiological↗
PubMed1999

Rhodopsin and phototransduction.

Recent studies on rhodopsin structure and function are reviewed and the properties of vertebrate as well as invertebrate rhodopsin described. Open issues such as the 'red shift' of the absorbance spectra are emphasized in the light of the present model of the retinal-binding pocket. The processes that restore the rhodopsin content in photoreceptors are also presented with a comparison between vertebrate and invertebrate visual systems. The central role of rhodopsin in the phototransduction cascade becomes evident by examining the main reports on light-activated conformational changes of rhodopsin and its interaction with transducin. Shut-off mechanisms are considered by reporting the studies on the sites of rhodopsin phosphorylation and arrestin binding. Furthermore, recent findings on the energetics of phototransduction point out that the ATP needed for photoreception in vertebrates is synthesized in the outer segments where phototransduction events take place.

Amino Acid Sequence↗
PubMed2002

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms.

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Positional cloning and molecular characterization of an immunodominant cytotoxic determinant of the mouse H3 minor histocompatibility complex.

Immune responses to minor histocompatibility antigens are poorly understood and present substantial barriers to successful solid tissue and bone marrow transplantation among MHC-matched individuals. We exploited a unique positional cloning approach relying on the potent negative selection capability of cytotoxic T cells to identify the H3a gene responsible for immunodominant H2-Db-restricted determinants of the classically defined mouse autosomal H3 complex. The allelic basis for reciprocal H3a antigens is two amino acid changes within a single nonamer H2-Db-binding peptide. The H3a gene, now called Zfp106, encodes a 1888-amino acid protein with three zinc fingers and a beta-transducin domain consistent with DNA/protein binding. A region of ZFP106 is identical to a 600-amino acid sequence implicated in the insulin receptor signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A molecular mechanism for the phosphorylation-dependent regulation of heterotrimeric G proteins by phosducin.

Visual signal transduction is a nearly noise-free process that is exquisitely well regulated over a wide dynamic range of light intensity. A key component in dark/light adaptation is phosducin, a phosphorylatable protein that modulates the amount of transducin heterotrimer (Gt alpha beta gamma) available through sequestration of the beta gamma subunits (Gt beta gamma). The structure of the phosphophosducin/Gt beta gamma complex combined with mutational and biophysical analysis provides a stereochemical mechanism for the regulation of the phosducin-Gt beta gamma interaction. Phosphorylation of serine 73 causes an order-to-disorder transition of a 20-residue stretch, including the phosphorylation site, by disrupting a helix-capping motif. This transition disrupts phosducin's interface with Gt beta gamma, leading to the release of unencumbered Gt beta gamma, which reassociates with the membrane and Gt alpha to form a signaling-competent Gt alpha beta gamma heterotrimer.

Animals↗
PubMed2003

Rhodopsin phosphorylation: 30 years later.

Phototransduction in vertebrate photoreceptor cells mediated by rhodopsin is one of the most comprehensively examined G protein-coupled receptor (GPCR) signaling pathways. The signal transduction pathway can be mapped from the initial absorption of light to conformational changes within rhodopsin, through activation of the G protein transducin, and to the ultimate closure of the cation cGMP-gated channels in the plasma membrane. Furthermore, phototransduction has become an intensely studied model system for understanding the desensitizing processes that allow reduced non-linear responses of photoreceptor cells to increasing levels of illumination. Although some general themes appear to occur in GPCR systems, the details of these desensitizing processes are likely to be specific to each of the receptors. These differences are attributed to the fact that each receptor has unique kinetic constraints, amplification levels, tolerance to basal constitutive activity, intracellular internalization and recycling, redundancy of isoforms, and morphologies of the cell of their expression. One of the biochemical processes that are believed to be a common part of this desensitization of the GPCR-mediated cascade is receptor phosphorylation catalyzed by members of a small family of the GPCR kinases. The enzymatic, physiological and genetic aspects of rhodopsin phosphorylation and rhodopsin kinase have been characterized extensively over the last 30 yr. However, new structurally based approaches to examining rhodopsin kinase and rhodopsin phosphorylation are still awaiting further investigations. We present here a summary of the current understanding of rhodopsin phosphorylation and the properties of rhodopsin kinase, along with some expectations of future investigations into these topics.

Animals↗
PubMed1991

Dim background light and Cerenkov radiation from 32P block reversal of rhodopsin phosphorylation in intact frog retinal rods.

The phosphorylation of photoexcited rhodopsin (Rho*) is thought to inactivate this receptor by inhibiting its interaction with the GTP-binding protein transducin (Gt). Here we report that the time course of phosphorylation-dephosphorylation after bright illumination of intact rod outer and inner segments (ROS-RIS) incubated in 33Pi can be altered if the ROS-RIS are first exposed to levels of dim illumination that cause light adaptation in these ROS-RIS. The dephosphorylation of greater than 10(7) phosphorylated rhodopsin molecules/ROS following a bright flash can be blocked by prior dim continuous illumination (generating 10(3) Rho*/ROS/s) that cumulatively bleaches approximately 10(5) rhodopsin molecules/ROS. The phenomenon has not been previously noted because these low levels of light are emitted as a result of Cerenkov radiation from the 32P isotope that is usually employed to monitor rhodopsin phosphorylation. The inhibition of rhodopsin dephosphorylation by dim conditioning illumination is observed in intact ROS-RIS but is lost when ROS-RIS are electropermeabilized or fragmented.

Animals↗
PubMedDate not supplied

Co-expression of murine opsins facilitates identifying the site of cone adaptation.

Murine cones contain two opsins in the same cone, one ultraviolet (UV) and the other middle-wavelength sensitive (M). A long-wavelength flash only affecting M-opsin suppresses the cone electroretinogram (ERG) produced by light absorption of UV-cone opsin raising the hypothesis that activation of M-cone opsin suppresses UV-cone opsin responses in the same cone. Here we show that pharmacologic blockade of synaptic transmission in the superfused murine retina, which eliminates interaction from second-order neurons, fails to prevent suppression of the UV-opsin driven pathway by long-wavelength stimuli. This proves that the antagonism must be occurring in the same cone, co-expressing both opsins. Our results show that UV-opsin suppression successively ceases in presence of the M-opsin activating background light, which implies that cone light adaptation is controlled at the opsin stage, before activation of transducin. It also reveals the time course of a transient desensitization of cones due to post-opsin factors in the transduction cascade.

Adaptation, Ocular↗
PubMedDate not supplied

Conditioning light differentially desensitizes rod phototransduction mediated by native and 9-demethyl analog visual pigment.

Light adaptation in rod photoreceptors is thought to involve down-regulation of the signaling activity of photoactivated rhodopsin (R*). However, electrophysiological evidence in support of this notion has come largely from studies of truncated, perfused rod outer segments and of rods genetically engineered to perturb known steps in R* deactivation. To test this hypothesis within intact native rods, we examined the effect of a fixed conditioning flash on rods prepared to contain 9-demethyl rhodopsin (9dR) in addition to residual rhodopsin. 9dR, an opsin-based photopigment containing 11-cis 9-demethylretinal as its chromophore, exhibits a blue-shifted excitation spectrum and sluggish deactivation kinetics, properties that distinguish the signaling activities of photoactivated 9dR (9dR*) from those of R*. Saturating photocurrent responses mediated preferentially by R* and 9dR* were obtained with test flash stimulation at 640 and 440 nm, respectively, under dark-adapted conditions (unconditioned response) and at a fixed time after a 640-nm conditioning flash of fixed high intensity. At each test wavelength, the decrease in photocurrent saturation period induced by the conditioning flash was analyzed to determine psi, the sensitivity of the conditioned response relative that of the unconditioned response; psi640/psi440, the ratio of relative sensitivities, was then obtained. Data obtained from 12 rods yielded psi640/psi440 = 0.60 +/- 0.13 (mean +/- SD). As common pools of transducin and other downstream components mediate transduction initiated by both R* and 9dR*, the finding that psi640 < psi440 provides direct evidence for the down-regulation specifically of R*'s signaling activity during rod light adaptation.

Adaptation, Ocular↗
PubMedDate not supplied

Molecular genetics of color-vision deficiencies.

The normal X-chromosome-linked color-vision gene array is composed of a single long-wave-sensitive (L-) pigment gene followed by one or more middle-wave-sensitive (M-) pigment genes. The expression of these genes to form L- or M-cones is controlled by the proximal promoter and by the locus control region. The high degree of homology between the L- and M-pigment genes predisposed them to unequal recombination, leading to gene deletion or the formation of L/M hybrid genes that explain the majority of the common red-green color-vision deficiencies. Hybrid genes encode a variety of L-like or M-like pigments. Analysis of the gene order in arrays of normal and deutan subjects indicates that only the two most proximal genes of the array contribute to the color-vision phenotype. This is supported by the observation that only the first two genes of the array are expressed in the human retina. The severity of the color-vision defect is roughly related to the difference in absorption maxima (lambda(max)) between the photopigments encoded by the first two genes of the array. A single amino acid polymorphism (Ser180Ala) in the L pigment accounts for the subtle difference in normal color vision and influences the severity of red-green color-vision deficiency. Blue-cone monochromacy is a rare disorder that involves absence of L- and M-cone function. It is caused either by deletion of a critical region that regulates expression of the L/M gene array, or by mutations that inactivate the L- and M-pigment genes. Total color blindness is another rare disease that involves complete absence of all cone function. A number of mutants in the genes encoding the cone-specific alpha- and beta-subunits of the cGMP-gated cation channel as well as in the alpha-subunit of transducin have been implicated in this disorder.

Animals↗
PubMedDate not supplied

Subcellular localization of phosducin in rod photoreceptors.

Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the beta,gamma subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

Animals↗
PubMedDate not supplied

Toward a unified model of vertebrate rod phototransduction.

Recently, we introduced a phototransduction model that was able to account for the reproducibility of vertebrate rod single-photon responses (SPRs) (Hamer et al., 2003). The model was able to reproduce SPR statistics by means of stochastic activation and inactivation of rhodopsin (R*), transducin (G alpha ), and phosphodiesterase (PDE). The features needed to capture the SPR statistics were (1) multiple steps of R* inactivation by means of multiple phosphorylations (followed by arrestin capping) and (2) phosphorylation dependence of the affinity between R* and the three molecules competing to bind with R* (G alpha, arrestin, and rhodopsin kinase). The model was also able to account for several other rod response features in the dim-flash regime, including SPRs obtained from rods in which various elements of the cascade have been genetically disabled or disrupted. However, the model was not tested under high light-level conditions. We sought to evaluate the extent to which the multiple phosphorylation model could simultaneously account for single-photon response behavior, as well as responses to high light levels causing complete response saturation and/or significant light adaptation (LA). To date no single model, with one set of parameters, has been able to do this. Dim-flash responses and statistics were simulated using a hybrid stochastic/deterministic model and Monte-Carlo methods as in Hamer et al. (2003). A dark-adapted flash series, and stimulus paradigms from the literature eliciting various degrees of light adaptation (LA), were simulated using a full differential equation version of the model that included the addition of Ca2+-feedback onto rhodopsin kinase via recoverin. With this model, using a single set of parameters, we attempted to account for (1) SPR waveforms and statistics (as in Hamer et al., 2003); (2) a full dark-adapted flash-response series, from dim flash to saturating, bright flash levels, from a toad rod; (3) steady-state LA responses, including LA circulating current (as in Koutalos et al., 1995) and LA flash sensitivity measured in rods from four species; (4) step responses from newt rods ( Forti et al., 1989) over a large dynamic range; (5) dynamic LA responses, such as the step-flash paradigm of Fain et al. (1989), and the two-flash paradigm of Murnick and Lamb (1996); and (6) the salient response features from four knockout rod preparations. The model was able to meet this stringent test, accounting for almost all the salient qualitative, and many quantitative features, of the responses across this broad array of stimulus conditions, including SPR reproducibility. The model promises to be useful in testing hypotheses regarding both normal and abnormal photoreceptor function, and is a good starting point for development of a full-range model of cone phototransduction. Informative limitations of the model are also discussed.

Adaptation, Ocular↗
PubMedDate not supplied

Does rod phototransduction involve the delayed transition of activated rhodopsin to a second, more active catalytic state?

Recovery kinetics of the saturating photocurrent response in amphibian rods suggest regulation of the visual signal by a first-order deactivation reaction with an exponential time constant (tau(c)) of about 2 s. The original hypothesis that tau(c) represents the lifetime of activated rhodopsin (R*) in a single-step deactivation appears at odds with several recent findings, for example, that Ca2+, a known regulator of the enzymatic phosphorylation of R*, does not regulate the value of tau(c). A recently proposed alternative hypothesis, that tau(c) is the lifetime of activated transducin and that the R* lifetime is relatively short (approximately 0.4 s), appears consistent with the Ca2+ data but is difficult to reconcile with a high specific catalytic activity of R*. The present theoretical study proposes a rate-equation model of R* activation and deactivation in amphibian rods that is generally consistent with observed properties of the tau(c)-associated reaction and the action of Ca2+ as well as with the stereotyped nature of the single-photon response. The model is developed by considering the effect of background light on a time-dependent variable, R*eff, defined as the effective total level of R* activity. Central starting assumptions are that Ca2+ reduction mediates the effect of background light on R*eff(t) and that background desensitization of the photocurrent flash response derives from this action of Ca2+. Construction of the model is guided by criteria based on previous experimental findings. Among these are the approximate constancy of background desensitization expressed at near-peak and later times in the flash response, and the large (approximately 10-fold) dynamic range of this desensitization. The proposed model hypothesizes that an event regulated by Ca2+ feedback causes activated rhodopsin to become susceptible to a two-phase, stochastic deactivation process, the second phase of which is characterized by tau(c). A central prediction of the model is the regulated transition of flash-activated R* to "R**", a state exhibiting greatly increased catalytic activity.

Adaptation, Physiological↗
PubMed2000

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function.

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Amino Acid Sequence↗
PubMed2000

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo.

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.

Antiviral Agents↗
PubMed2006

Surface plasmon resonance study of g protein/receptor coupling in a lipid bilayer-free system.

Surface plasmon resonance (SPR) spectroscopy is a technique to study protein-protein interactions in real time; however, application of SPR spectroscopy for investigations of membrane receptors is difficult with respect to functional and uniform immobilization of receptors on a biosensor surface. In the current study, we developed a simple, direct, biosensor-based approach to monitor the molecular interactions between G protein transducin (Gt) and rhodopsin (Rho), a prototypical G protein-coupled receptor (GPCR). Detergent-solubilized dark-adapted Rho was captured onto a biosensor surface via lectin interaction, enabling site-directed immobilization of the receptor that made its cytoplasmic surface accessible to a coupling G protein. The system resembled the natural system with respect to receptor density, binding of Gt following flash or constant light application, fast GTP-dependent dissociation of Gt from Rho, regeneration of Rho, and dependence of Gt binding on light intensity and on concentration of Gt. The apparent KD of the Gt/Rho interaction was 13.6 nM. Our results validate the use of SPR spectroscopy as a tool to study G protein activation in GPCR systems and could be extended for application to other interaction partners of GPCRs.

Animals↗