PHARMACOLOGY

Ribbon drawings of rhodopsin.
(A) Parallel to the plane of the membrane (stereoview). A view into the membrane plane is seen from the cytoplasmic
(B)and intradiscal side (C) of the membrane.
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M.
Crystal structure of rhodopsin: A G protein-coupled receptor.Science 2000 Aug 4 289:5480 739-45

 

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Back to top Double labelling with DIG labelled GCAP3 cRNA (blue) with anti-cone opsins antibodies (red). (A) Double labelling with anti-blue cone opsin. Anti-blue cone opsin labels cone photoreceptors which hybridizes GCAP3 cRNA probe (blue). (B) Double labelling with anti-red/green cone opsin. Red/green cones are labelled with GCAP3 cRNA. Scale bar, 50 mm

Imanishi Y, Li N, Sokal I, Sowa ME, Lichtarge O, Wensel TG, Saperstein DA, Baehr W, Palczewski K: Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man. Eur J Neurosci 2002;15: 63-78.
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Evolutionary trace analysis of the GCAP and NCBP families. An Evolutionary trace analysis (ET) was performed on both the GCAP family (33 members) and the NCBP family (106 members, including the GCAP subfamily). The results were mapped onto the structure of Ca2+-bound GCAP2 (Ames et al., 1999). EF hands, class specific, and invariant residues are coloured according to the colour table, with numbering and amino acid designation based on bovine GCAP1. Views A and B are rotated 180° about the y-axis with respect to each other as are C and D. (A, B) ET analysis of the GCAP subfamily reveals a large surface cluster of both class specific and invariant residues from EF1 and EF2. This site is much smaller in size for the NCBP trace suggesting that this region is specific for the GCAP subfamily (A vs. C, B vs. D). The stretch of ET-identi®ed surface residues beginning with Phe73 and ending with Phe135 (A vs. C) is found in both the NCBP and the GCAP traces, indicating that this site is important for the functional specificity of the entire NCBP family.

Imanishi Y, Li N, Sokal I, Sowa ME, Lichtarge O, Wensel TG, Saperstein DA, Baehr W, Palczewski K: Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man. Eur J Neurosci 2002;15: 63-78.
Back to top Organization of the mouse retina and in situ localization of CaBP5 and CaBP8 transcripts. (A) Light micrograph of the mouse retina. ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer containing rod and cone bipolar cells; IPL, inner plexiform layer containing amacrine, horizontal and glia cells; GCL, ganglion cell layer. (B) In situ hybridization analysis of CaBP5 expression in the mouse retina. Subpopulation of inner nuclear cells showed hybridization signals (arrows) consistent with the previous immunocytochemical report (61). (C) In situ hybridization analysis of CaBP8 expression in the mouse retina. Subpopulation of inner nuclear cells showed hybridization signals (arrows). The expression pattern was different from CaBP5.

Haeseleer F, Imanishi Y, Sokal I, Filipek S, Palczewski K: Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man. Eur J Neurosci 2002;15: 63-78.
Back to top The environment of the 11-cis-retinal chromophore. (A) Experimental electron density of 3.3 Å resolution with the final model of 3.3 Å data set using MAD phases after NCS averaging and solvent flattening with DM (20) for the retinal chromophore. Blue for 2|Fo| - |Fc| map (1). (B) Electron density for the retinal chromophore with the current model refined against the 2.8 Å data set. Blue for 2|Fo| - |Fc| map (1) and red for omit |Fo| - |Fc| map (5) phases calculated using the current model. (C) Schematic showing the side chains surrounding the 11-cis-retinylidene group, viewed from cytoplasmic side. Ala169 interacts with -ionone ring of all-trans-retinal in photo-activated states (58). When the intrinsic 11-cis-retinal was substituted by all-trans-retinal in the crystal structure, the -ionone ring can reach Ala169 residue. (D) Schematic presenting the residues within 4.5 Å distance from retinal molecule. Blue labels indicate the distances between Schiff base nitrogen atom and charged or polar atoms within 4.5 Å.

Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M, Crystal structure of rhodopsin: A G protein-coupled receptor.Science 2000 Aug 4 289:5480 739-45
Back to top Permeability of rod plasma membranes containing the Zn2+-sensitive form of the -toxin pore (H5K8A) demonstrated by confocal fluorescence localization of low molecular weight intracellular tracer. Retinal punches were incubated in the presence (A and B) or absence (C and D) of -toxin. The addition of Zn2+ to the incubation medium causes pore closure (B and D). The low molecular weight intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride (Neurobiotin) was added to all samples to assess permeability of -toxin pores under various conditions. An -toxin-specific polyclonal antibody was used to immunolocalize this protein (green). -Toxin is restricted to the plasma membrane-surrounding rod inner segments (IS) and outer segments (OS) of treated retinas (A and B). Streptavidin-Cy3 was used to localize neurobiotin in these samples (red). Neurobiotin is present in the cytoplasm of rod OS and IS (A), confirming that -toxin forms functional pores in these cells. Neurobiotin is prevented from diffusing through -toxin pores closed by Zn2+ (B).

Otto_Bruc AE, Fariss RN, Van Hooser JP, Palczewski K, Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin., Proc Natl Acad Sci U S A 1998 Dec 8 95:25 15014-9
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Environments of 6 Trp residues in native GCAP1 and its Trp mutants. The ribbon representation of the protein is colored as follows: EF-hands 1 through 4 in yellow, wild-type Trp residues in green, new Trp residues introduced by mutation in red, and the rest of the protein in purple. The model was generated as described under "Material and Methods."

Sokal I, Otto_Bruc AE, Surgucheva I, Verlinde CL, Wang CK, Baehr W, Palczewski K, Conformational changes in guanylyl cyclase-activating protein 1(GCAP1) and its tryptophan mutants as a function of calcium concentration., J Biol Chem 1999 Jul 9 274:28,19829-37
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Western blot analysis of the expression of GCAPs in human and bovine retina using specific anti-GCAP antibodies. A, specificity of anti-GCAP antibodies. Anti-GCAP sera were depleted from cross-reactive IgGs and purified as described under "Materials and Methods." Lane 1, purified GCAP1 (1 µg); lane 2, purified GCAP2 (1 µg); lane 3, purified GCAP3 (1 µg). a, SDS-polyacrylamide gel stained with Coomassie Brilliant Blue R-250. Lane S, standard proteins (94, 67, 43, 30, 20, and 14 kDa); b-d, reactivity of GCAP1/GCAP2/GCAP3 by Western blot analysis (loaded as in a) with anti-GCAP1 (UW14), anti-GCAP2 (UW50) and anti-GCAP3 (UW84), respectively. Lane S, standard proteins (104, 81, 47.7, 34.6, 28.3, and 19.2 kDa). B, specificity of anti-GCAP antibodies tested by enzyme-linked immunosorbent assay. a, anti-GCAP1. UW14 antibody (0.45 µg) and increasing amounts of GCAPs were added to GCAP1-coated plates. GCAP2 and GCAP3 had no effect, and for GCAP1, IC50 = 0.09 ± 0.02 µg; b, anti-GCAP2 antibodies; UW50 antibodies (0.45 µg) and increasing amounts of GCAPs were added to GCAP2-coated plates. GCAP1 and GCAP3 had no effect, and for GCAP2, IC50 = 0.10 ± 0.02 µg; c, anti-GCAP3. UW84 antibodies (0.5 µg) and increasing amounts of GCAPs were added to GCAP3-coated plates. GCAP1 and GCAP2 had no effect, and for GCAP3, IC50 = 0.25 ± 0.04 µg. C, Western blot analysis of the expression of GCAPs in bovine and human retina. Human (H) or bovine (B) retinal extracts (~8 µg each) were probed with anti-GCAP1 (UW14), anti-GCAP2 (UW50), anti-GCAP3 (UW84), or anti-GCAP3 peptide (UW87).

Haeseleer F, Sokal I, Li N, Pettenati M, Rao N, Bronson D, Wechter R, Baehr W, Palczewski K, Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily., J Biol Chem 1999 Mar 5 274:10 6526-35
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Expression and purification of human 11-cis-RDH. A, 11-cis-RDH containing His6 tag at the C terminus was expressed in Sf9 insect cells and purified employing Ni2+ column chromatography in Genapol ("Materials and Methods"). The enzyme was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (lane a) and identified by Western blotting with an anti-11-cis-RDH-specific antibody (10)(lane b). B, assays of 11-cis-RDH activity. The assays were carried out using phase partition assay with pro-S-[4-3H]NADH and 11-cis-retinal as substrates, 11-cis-RDH from Sf9 insect cell membranes, HEK293 cell membranes, and purified 11-cis-RDH served as the source of enzyme.

Jang GF, McBee JK, Alekseev AM, Haeseleer F, Palczewski K, Stereoisomeric specificity of the retinoid cycle in the vertebrate retina.,J Biol Chem 2000 Sep 8 275:36 28128-38
Back to top Immunolocalization of CaBP1 and CaBP5 in mouse retina. CaBP1 (UW72) (A) and CaBP5 (UW89) (B) expression is prominent in cell bodies located in the INL. CaBP1 expression is restricted to a small subset of INL neurons, while CaBP5 is expressed by a majority of INL neurons. No immunolabeling is visible in sections of mouse retina incubated in pre-immune sera from UW72 (C) or UW89 (D). Preincubation of UW72 sera with purified CaBP1 (600 nM) abolishes CaBP1 immunolabeling (E).Preincubation of UW89 sera with purified CaBP5 (600 nM) abolishes CaBP5 immunolabeling (F). Sections of mouse retina were double-labeled with antibodies to PKC and either CaBP1 (G) or CaBP5 (H). PKC immunolabels rod bipolar cell bodies and their processes (green). Very few CaBP1-labeled cells (red) are PKC-positive. In contrast, most CaBP5-positive cells (red) are double-labeled by antibodies to PKC (arrowheads, H), confirming their identity as rod bipolar cells. Cone bipolars are labeled by antibodies to CaBP5 but not PKC. Magnification bar: A-F, 50 µm; G-H, 25 µm.

Haeseleer F, Sokal I, Verlinde CL, Erdjument_Bromage H, Tempst P, Pronin AN, Benovic JL, Fariss RN, Palczewski K, Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin., J Biol Chem 2000 Jan 14 275:2 1247-60
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Primary structure of CaBPs and phylogenetic tree. A, sequence alignment of h-CaBP1, h-CaBP2, h-CaBP3, h-CaBP4, h-CaBP5, and h-CaM. Alignment of the deduced aa sequences of h-CaBPs with CaM. The identical residues in all sequences are shown in white letters on black background. The conservative substitutions (E=D; S=T; V=M=I=L; K=R) are shown in white letters on dark gray background. The residues that are identical among CaBPs are shaded in light gray. Functional EF-hand motifs are shown in shaded boxes, and the nonfunctional EF-hand motif is in an open box. The asterisk represents the myristoylation site, and the down arrowhead over a solid bar indicates a 4-amino acid-long extension of the central -helix. Arrows indicate the intron/exon junctions for the h-CaBP1, 2, 4, 5, and CaM genes. Vertical bars indicate the intron/exon junctions in CaBP3 and in alternative splice forms of CaBP1 and CaBP2. L- indicates the long spliced forms of CaBPs, and the underlined aa residues are absent in the short spliced forms. The letters h and s above the sequences indicate -helices and -strands, respectively. B, phylogenetic tree. The tree was built with a bootstrap analysis of neighbor-joining distance using PAUPSearch in GCG (Genetics Computer Group). The sequences included, with their GenBankTM/EMBL accession numbers in parentheses, are: h-CaM (A31920); h-CaM-like protein (P27482); rat caldendrin (Y17048); human recoverin (S62028); GCAP1 (L36859); GCAP2 (see Ref. 30); GCAP3 (AF110002); chicken visinin (P22728); bovine neurocalcin (JH0616); VILIP1: human visinin-like protein 1 (U14747); VILIP2: rat visinin-like protein 2 (P35332); VILIP3: human visinin-like protein 3 (P37235); NCS1: rat neuronal Ca2+ sensor 1 (P36610); CaBP4 (unpublished sequence); CaBP1, CaBP2, CaBP3, and CaBP5 are novel sequences reported in this study.

Haeseleer F, Sokal I, Verlinde CL, Erdjument_Bromage H, Tempst P, Pronin AN, Benovic JL, Fariss RN, Palczewski K, Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin., J Biol Chem 2000 Jan 14 275:2 1247-60
Back to top Drawing of the vertebrate rod photoreceptor cell. This is a highly differentiated post-mitotic cell composed of a sac of internal membranes, called rod outer segment, containing disks, enveloped by the plasma membranes. The ROS is connected to the inner segment by a tiny cilium. This fragile connection allows the ROS to be easily separated and purified by density flotation methods. The most abundant protein of ROS is rhodopsin (> 90%). Bovine retina was a source of highly purified rhodopsin for the crystallographic studies. The rod cell is highly sensitive and can detect a single photon, but saturates at low light intensity (˜104 photons/s). To support such high sensitivity, rhodopsin molecules (108) must be inactive. The spontaneous thermal activation of rhodopsin occurs with a half life of ˜ 400 years. This property implies that rhodopsin must be constrained in the inactive conformation by multiple mechanisms, each of low probability. Once photoactivated, the 11-cis-retinylidene chromophore isomerizes to all-trans-retinylidene and causes propagation and amplification of the signal, activating hundreds of transducin molecules.

Slawomir Fikipek, Ronald E. Stenkamp, David C. Teller, Krzysztof Palczewski: G Protein-Coupled Receptor Rhodospin: A Prospectus. Annu Rev Physiol 2003;65:851-879 (Supplemental Materials)
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