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ABSTRACT: In diabetic retinopathy, vision loss is usually secondary to macular edema, which is thought to depend on the functional integrity of the blood-retina barrier. The levels of advanced glycation end-products in the vitreous correlate with the progression of diabetic retinopathy. Natriuretic peptides (NP) are expressed in the eye and their receptors are present in the RPE. Here, we investigated the effect of glycated-albumin (Glyc-alb), an AGE model, on RPE barrier function and the ability of NP to suppress this response. Transepithelial electrical resistance (TEER) measurements were utilized to assess the barrier function of ARPE-19 and human fetal RPE monolayers. The monolayers were treated with 0.1 μg/mL to 100 μg/mL Glyc-alb in the absence or presence of 1 pM to 100 nM of apical ANP, BNP or CNP. Glyc-alb induced a significant reduction in TEER within two hours. This response was concentration-dependent (EC(50)=2.3 μg/mL) with a maximal reduction of 40±2% for ARPE-19 and 27±7% for hfRPE at 100 μg/mL six hours post treatment. One hour pretreatment with ANP, BNP or CNP blocked the reduction in TEER induced by Glyc-alb (100 μg/mL). The suppression of the Glyc-alb response by NP was dependent upon the generation of cGMP and exhibited a rank order of agonist potency consistent with the activation of NPR2 subtype (CNP>BNP≥ANP). Our data demonstrate that Glyc-alb is effective in reducing RPE barrier function and this response is suppressed by NP. Moreover, these studies support the idea that NPR2 agonists can be potential candidates for treating retinal edema in diabetic patients.
Journal of Pharmacology and Experimental Therapeutics 10/2012; · 3.83 Impact Factor
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ABSTRACT: The retinoid cycle is a series of biochemical reactions within the eye that is responsible for synthesizing the chromophore, 11-cis retinal, for visual function. The chromophore is bound to G-protein coupled receptors, opsins, within rod and cone photoreceptor cells forming the photosensitive visual pigments. Integral to the sustained function of photoreceptors is the continuous generation of chromophore by the retinoid cycle through two separate processes, one that supplies both rods and cones and another that exclusively supplies cones. Recent findings such as RPE65 localization within cones and the pattern of distribution of retinoid metabolites within mouse and human retinas have challenged previous proposed schemes. This review will focus on recent findings regarding the transport of retinoids, the mechanisms by which chromophore is supplied to both rods and cones, and the metabolism of retinoids within the posterior segment of the eye.
Progress in Retinal and Eye Research 10/2012; · 9.45 Impact Factor
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ABSTRACT: The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.
Journal of Biological Chemistry 05/2012; 287(26):22276-86. · 4.77 Impact Factor
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ABSTRACT: Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N-retinylidene-N-retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl(-/-) mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl(-/-) retina contain only cone-like pigments, and produce cone-like responses to photostimulation. The cone-like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl(-/-) mice were similar to those from wild type mice, with λ(max) ca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.
Photochemistry and Photobiology 03/2012; · 2.41 Impact Factor
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Advances in experimental medicine and biology 01/2012; 723:75-81. · 1.09 Impact Factor
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ABSTRACT: The two most commonly used in vitro models of the retinal pigment epithelium (RPE) are fetal human RPE (fhRPE) and ARPE-19 cells; however, studies of their barrier properties have produced contradictory results. To compare their utility as RPE models, their morphologic and functional characteristics were analyzed.
Monolayers of both cell types were grown on permeable membrane filters. Barrier function and cellular morphology were assessed by transepithelial resistance (TER) measurements and immunohistochemistry. Protein expression was evaluated by immunoblotting and ELISA assays, and retinoid metabolism characterized by HPLC.
Both cultures developed tight junctions. However, only the fhRPE cells were pigmented, uniform in size and shape, expressed high levels of RPE markers, metabolized all-trans retinal, and developed high TER (>400 Ωcm(2)). The net secretion of pigment-epithelium-derived factor (PEDF) was directed apically in both cultures, but fhRPE cells exhibited secretion rates a thousand-fold greater than in ARPE-19 cells. The net secretion of vascular endothelial growth factor (VEGF) was significantly higher in fhRPE cultures and the direction of this secretion was basolateral; while net secretion was apical in ARPE-19 cells. In fresh media, VEGF-E reduced TER in both cultures; however, in conditioned media fhRPE cells did not respond to VEGF-E administration, but retreatment of the conditioned media with anti-PEDF antibodies allowed fhRPE cells to fully respond to VEGF-E.
Properties of fhRPE cells align with a functionally normal RPE in vivo, while ARPE-19 cells resemble a pathologic or aged RPE. These results suggest a utility for both cell types in understanding distinct, particular aspects of RPE function.
Investigative ophthalmology & visual science 09/2011; 52(12):8614-20. · 3.43 Impact Factor
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ABSTRACT: The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.
Age 12/2010; 33(4):543-54. · 6.28 Impact Factor
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ABSTRACT: Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometric method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopy overestimates the amount of A2E. This mass spectrometric approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.
Photochemical and Photobiological Sciences 10/2010; 9(11):1513-9. · 2.58 Impact Factor
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ABSTRACT: In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.
Journal of Biological Chemistry 05/2010; 285(28):21837-48. · 4.77 Impact Factor
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ABSTRACT: In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with
cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional upregulation of
cardiac α- and β-tubulin and microtubule stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule
decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Since the major determinant of this affinity is site-specific
MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of
each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied
feline myocardium; volume-overloaded myocardium having an equal degree and duration of hypertrophy but normal functional and
cytoskeletal properties served as a control for any non-specific growth-related effects. After cloning cDNA encoding feline
MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation.
Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser472 in the MAP4
amino-terminal projection domain and at Ser924 and Ser1056 in the assembly-promoting region of the carboxyl-terminal microtubule-binding
domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type
and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed
in normal cardiocytes following an equivalent level of MAP4 expression. The Ser924Ala MAP4 mutant produced a microtubule phenotype
indistinguishable from that seen in pressure overload hypertrophy, such that Ser924 MAP4 dephosphorylation during pressure
overload hypertrophy may be central to this cytoskeletal abnormality.
Journal of Biological Chemistry 04/2010; · 4.77 Impact Factor
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ABSTRACT: Amyloid-beta (Abeta) accumulates in several types of retinal degeneration and in Alzheimer's disease (AD), but its source has been unclear. We detected the neuronal 695 amino acid form of amyloid-beta protein precursor (AbetaPP) in the normal retina and AbetaPP751 in the retinal pigment epithelium (RPE) and anterior eye tissues. Similar to the brain, alpha- and beta-secretases cleaved AbetaPP to soluble derivatives (sAbetaPP) alpha or beta and membrane-bound C-terminal fragments alpha or beta in the retina and RPE. Levels of sAbetaPP were particularly high in the vitreous and low in aqueous humor revealing a molecular barrier for AbetaPP. In contrast, Abeta40 and Abeta42 levels were only 50% lower in the aqueous than the vitreous humor, indicating relatively barrier-free movement of Abeta. These studies demonstrated a relatively high yield of AbetaPP and Abeta in the ocular fluids, which may serve as a trackable marker for AD. In addition, failure of free clearance from the eye may trigger retina degeneration in a manner similar to Abeta-related neurodegeneration in AD.
Journal of Alzheimer's disease: JAD 04/2010; 20(4):1243-53. · 3.74 Impact Factor
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ABSTRACT: Wet age-related macular degeneration (AMD) attacks the integrity of the retinal pigment epithelium (RPE) barrier system. The pathogenic process was hypothesized to be mediated by vascular endothelial growth factor (VEGF) and antagonized by pigment epithelium-derived factor (PEDF). To dissect these functional interactions, monolayer cultures of RPE cells were established, and changes in transepithelial resistance were evaluated after administration of PEDF, placenta growth factor (VEGF-R1 agonist), and VEGF-E (VEGF-R2 agonist). A recently described mechanism of VEGF inhibition in endothelia required the release of VEGF-R1 intracellular domain by gamma-secretase. To evaluate this pathway in the RPE, cells were pretreated with inhibitors DAPT or LY411575. Processing of VEGF receptors was assessed by Western blot analysis. Administration of VEGF-E rapidly increased RPE permeability, and PEDF inhibited the VEGF-E response dose-dependently. Both gamma-secretase antagonists prevented the inhibitory effects of PEDF. The co-administration of PEDF and VEGF-E depleted the amount of VEGF-R2 in the membrane and increased the amount of VEGF-R2 ectodomain in the media. Therefore, the inhibitory effect of PEDF appears to be mediated via the processing of VEGF-R2 by gamma-secretase. gamma-Secretase generates the amyloid-beta (Abeta) peptide of Alzheimer disease from its precursor (amyloid precursor protein). This peptide is also a component of drusen in dry AMD. The results support the hypothesis that misregulation of gamma-secretase may not only lead to Abeta deposits in dry AMD but can also be damaging to RPE function by blocking the protective effects of PEDF to prevent VEGF from driving the dry to wet AMD transition.
Journal of Biological Chemistry 10/2009; 284(44):30177-86. · 4.77 Impact Factor
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ABSTRACT: Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.
Journal of Biological Chemistry 05/2009; 284(25):16939-47. · 4.77 Impact Factor
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ABSTRACT: RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.
Journal of Biological Chemistry 01/2009; 284(5):3211-8. · 4.77 Impact Factor
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ABSTRACT: Rhodopsin (the photosensitive rod visual pigment) has been a model for photobiologic studies of the opsins as well as a structural model for G-protein-coupled receptors. The two palmitate groups attached to cysteines 322 and 323 are thought to serve as membrane anchors for the rhodopsin C-terminus, but the absence of the palmitates does not alter membrane localization. However, removal of the palmitates affects rhodopsin function. Therefore, it is important to quantitate the stability of rhodopsin palmitates to hydroxylamine, which is a widely utilized reagent in biochemical preparations of the apoprotein. We have developed a mass spectrometric method to quantitate the resulting opsin palmitylation. Our data show that both of the bovine rhodopsin palmitates are labile to hydroxylamine, with significant depalmitylation occurring at concentrations of >or=100 mM, with an EC(50) of 220 mM L(-1). The palmitate at position 322 is the more stable to hydroxylamine. Samples prepared in the presence of >50 mM should therefore be considered to be at least partially depalmitylated and the results interpreted accordingly.
Photochemistry and Photobiology 04/2008; 84(4):949-55. · 2.41 Impact Factor
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ABSTRACT: Fluid accumulation into the subretinal space and the development of macular edema is a common condition in age-related macular degeneration, diabetic retinopathy, and following ocular surgery, or injury. Vascular endothelial growth factor (VEGF) and other cytokines have been implicated in the disruption of retinal pigment epithelium (RPE) barrier function and a reduction in the regulated removal of subretinal fluid; however, the cellular and molecular events linking these agents to the disruption of barrier function have not been established. In the current study, cultures of ARPE-19 and primary porcine retinal pigment epithelium (RPE) cells were utilized to investigate the effects of the VEGF-induced modifications to the barrier properties of the RPE. The barrier function was determined by transepithelial resistance (TER) measurements and morphology of the RPE monolayers. In both ARPE-19 and primary porcine RPE cells the administration of VEGF produced a significant drop in TER, and this response was only observed following apical administration. Maximum reduction in TER was reached 5h post VEGF administration. These responses were concentration-dependent with an EC(50) of 502pg/mL in ARPE-19 cells and 251pg/mL in primary porcine cells. In both ARPE-19 and primary RPE cells, the response to VEGF was blocked by pretreatment with the relatively selective VEGF-R2 antagonists, SU5416 or ZM323881, or the protein tyrosine kinase inhibitor, genistein. Administration of the relatively selective VEGF-R2 agonist, VEGF-E, also reduced TER in a concentration-dependent manner (EC(50) of 474pg/mL), while VEGF-R1 agonist, placental growth factor (PlGF), did not significantly alter the TER. Immunolocalization studies demonstrated that confluent monolayers exhibited continuous cell-to-cell ZO-1 protein contacts and apical localization of the VEGF-R2 receptors. These data provide evidence that the VEGF-induced breakdown of RPE barrier function is mediated by the activation of apically-oriented VEGF-R2 receptors. Thus, VEGF-mediated increases in RPE permeability are initiated by a rise in intraocular levels of VEGF.
Experimental Eye Research 01/2008; 85(6):762-71. · 3.26 Impact Factor
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ABSTRACT: Palmitylation is a widespread modification in G-protein-coupled receptors and often a dynamic process. In rhodopsins, palmitylation is static on C322/C323. Red/green (M/LWS) cone opsins have no cysteines at corresponding positions and no palmitylation. Blue (SWS2) cone opsins have a single corresponding cysteine and mass spectrometric analysis showed partial palmitylation of salamander SWS2 cone opsin. Ultraviolet (SWS1) cone opsins have one corresponding cysteine, but only unpalmitylated opsin was observed for mouse and salamander. The results show that the static palmitylation found on rhodopsin is not found on cone opsins and suggest the possibility of an unidentified role for opsin palmitylation in cones.
Vision Research 01/2007; 46(27):4493-501. · 2.41 Impact Factor
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ABSTRACT: Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.
Journal of Chromatography B 11/2005; 825(2):169-75. · 2.89 Impact Factor
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ABSTRACT: Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.
Journal of Biological Chemistry 08/2005; 280(26):24293-300. · 4.77 Impact Factor
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ABSTRACT: The interactions of rhodopsin and the alpha-subunit of transducin (G(t)) have been mapped using a surface modification "footprinting" approach in conjunction with mass spectrometric analysis employing a synthetic peptide corresponding to C-terminal residues 340-350 of the alpha-subunit of G(t), G(t)alpha(340-350). Membrane preparations of unactivated (Rh) and light-activated rhodopsin (Rh*), each in the presence or absence of G(t)alpha(340-350), were acetylated with the water-soluble reagent sulfosuccinimidyl acetate, and the extent of the acetylation was determined by mass spectrometry. By comparing the differences in acetylation among Rh, Rh*, and the Rh-G(t)alpha(340-350) and Rh*-G(t)alpha(340-350) complexes, we demonstrate that the surface exposure of the acetylation sites was reduced by the conformational change associated with light activation, and that binding of G(t)alpha(340-350) blocks acetylation sites on cytoplasmic loops 1, 2, and 4 of Rh*. In addition, we show evidence of interaction between the end of the C-terminal tail of rhodopsin and G(t)alpha in the unactivated state of rhodopsin.
Biochemistry 10/2004; 43(35):11153-62. · 3.42 Impact Factor
