Interaction of arrestin with enolase1 in photoreceptors.

PURPOSE: Arrestin is in disequilibrium in photoreceptors, translocating between inner and outer segments in response to light. The purpose of this project was to identify the cellular component with which arrestin associates in the dark-adapted retina. METHODS: Retinas were cross-linked with 2.5 mM dithiobis(succinimidylpropionate) (DSP), and arrestin-containing complexes purified by anion-exchange chromatography. Tandem mass spectrometric analysis was used to identify the protein components in the complex. Enolase localization in photoreceptors was assessed by immunohistochemistry. Confirmation of interacting components was performed using immunoprecipitation and surface plasmon resonance (SPR). Enolase activity was also assessed in the presence of arrestin1. RESULTS: In retinas treated with DSP, arrestin cross-linked in a 125-kDa complex. The principal components of this complex were arrestin1 and enolase1. Both arrestin1 and -4 were pulled down with enolase1 when enolase1 was immunoprecipitated. In the dark-adapted retina, enolase1 co-localized with arrestin1 in the inner segments and outer nuclear layer, but remained in the inner segments when arrestin1 translocated in response to light adaptation. SPR of purified arrestin1 and enolase1 demonstrated direct binding between arrestin1 and enolase1. Arrestin1 modulated the catalytic activity of enolase1, slowing it by as much as 24%. CONCLUSIONS: The results show that in the dark-adapted retina, arrestin1 and -4 interact with enolase1. The SPR data show that the interaction between arrestin1 and enolase1 was direct, not requiring a third element to form the complex. Arrestin1 slowed the catalytic activity of enolase1, suggesting that light-driven translocation of arrestin1 may modulate the metabolic activity of photoreceptors.

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4).

The maintenance of photoreceptor cell polarity is compromised by the rhodopsin mutations causing the human disease autosomal dominant retinitis pigmentosa. The severe form mutations occur in the C-terminal sorting signal of rhodopsin, VXPX-COOH. Here, we report that this sorting motif binds specifically to the small GTPase ARF4, a member of the ARF family of membrane budding and protein sorting regulators. The effects of blocking ARF4 action were functionally equivalent to the effects of blocking the rhodopsin C-terminal sorting signal. ARF4 was essential for the generation of post-Golgi carriers targeted to the rod outer segments of retinal photoreceptors. Thus, the severe retinitis pigmentosa alleles that affect the rhodopsin sorting signal interfere with interactions between ARF4 and rhodopsin, leading to aberrant trafficking and initiation of retinal degeneration.

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Cryptic MBP epitope 1-20 is inducing autoimmune anterior uveitis without EAE in Lewis rats.

Lewis rats immunized with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Although several cryptic epitopes of MBP have strong encephalitogenic and uveitogenic properties, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. In this study, we showed that acetylation of the N-terminal amino acid did not produce encephalitogenicity, did not enhance uveitogenicity, and did not improve T cell proliferation in Lewis rats. The cytokine production profile induced by MBP(1-20) immunization was consistent with a Th1 response. In MBP-injected rats and in peptide-injected rats, the frequency of the IFN-gamma-secreting cells in MBP(69-89)-stimulated T cells was significantly higher than the frequency of IFN-gamma-secreting cells in MBP(1-20)-stimulated T cells. However, similar numbers of IFN-gamma-producing specific cells were found in the eyes of MBP(69-89) and MBP(1-20) immunized rats. In these rats, the iris-infiltrating cells consisted of a much higher percentage of CD4(+) T cells expressing L-selectin (CD62L) than did those cells found in the spinal cord. The results demonstrate that MBP(1-20) is immunogenic and uveitogenic, although it induced only weak proliferation and weak Th1 reaction. The fact that T cells with the same specificity have different effects on target organs suggested that, in the eye and spinal cord, a distinct mechanism might mediate the recruitment of cells to these organs.