Intron positions in the GRK2/GRK3 clade of G protein-coupled receptor kinases are highly conserved in metazoan evolution.

<p>Human GRK2 and GRK3 are aligned to their orthologs from an insect (<i>Drosophila melanogaster</i>, Dm) and a primitive metazoan (<i>Trichoplax adhaerens</i>, Ta). The amino acids with codons either interrupted or immediately followed by an intron in the genome sequence are highlighted by yellow shading. Ten positions are conserved in all four genes, seven additional positions are conserved between human genes and at least one of the invertebrate orthologs, and only five non-conserved positions were identified.</p

Characteristic features of the C-termini of GRK(1/7)4/5/6 clade.

<p><b>A.</b> The C-terminal sequences of the kinases of GRKa/(1/7)4/5/6 clade were aligned. The most conserved parts of the putative amphipathic helix and putative palmitoylation sites are highlighted in blue (GRK5), yellow (GRK6), green (GRK4), light brown (invertebrate GRKa), or magenta (insect GRKa). Cysteines (potential palmitoylation sites) downstream of the helix are highlighted in the same color as the helix in corresponding branch. Residues conserved in the entire clade (DL at the beginning of the alignment) or in the helix in individual branches are shown in bold. Characteristic pairs of hydrophobic residues within the helix are underlined. Species abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033806#pone-0033806-g001" target="_blank">Fig. 1</a>. <b>B.</b> The structure of the amphipathic helix in the C-terminus of human GRK5 and putative helical structures of homologous elements in GRKa from <i>T. adherens</i>, <i>C. owczarzaki</i>, and <i>D. melanogaster</i>. Hydrophobic residues are shown in blue, positively charged in red. Note that the structure of this element in GRKa of <i>Drosophila</i> is less likely to be helical due to the presence of three pralines (shown in green), although this is not the case for all insect GRKas. In any case, a stretch of positive charges in this GRK can also serve as the membrane anchor. The sequence of this element in each species, with the numbers of the first and last residue at the beginning and the end, respectively, are shown above the helical diagrams.</p

Plekstrin homology domain in the C-termini of GRKb/2/3 clade.

<p>The C-terminal sequences of the kinases of GRKb/2/3 clade and three GRKs from single-cell non-metazoan species <i>Phytophtora infestans</i> (apicomplexa), <i>Albugo laibachii</i> (oomycete), and <i>Ectocarpus siliculosus</i> (brown alga). PH domain residue numbering is based on the human GRK2. Conserved elements of the secondary structure and corresponding consensus sequences are shown above the alignment. Conserved core hydrophobic residues are shown in bold; conserved hydrophobic residues outside of the core are shown in bold blue; putative elements of the secondary structure are highlighted in yellow; conserved in GRK family W and R are highlighted in blue; conserved negative charges are shown in green bold underlined. Human GRK2 and GRK3 sequences are shown in red and light blue, respectively. Note incomplete set of elements of the putative PH domain in GRKs from single-cell pre-metazoan species. Species abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033806#pone-0033806-g001" target="_blank">Fig. 1</a>.</p

Phylogeny of the kinase domains of GRK proteins.

<p>Bootstrap support (in percent) of each partition is indicated by the numbers at the internal branches. The NCBI GI numbers are given for each sequence. The symbols next to the species name indicate the type of the C-terminal structure found in each GRK sequence: red squares indicate C-terminal PH domains; blue circles indicate C-terminal prenylation motifs: geranylgeranylation (solid circle) or farnesylation (open circle); and green triangles indicate either predicted canonical (solid triangle) or putative modified (open triangle) C-terminal amphipathic helix. Note that the C-terminal motives generally group with the GRK families. Thus, GRKb/2/3 family possesses C-terminal PH domain in most species including unicellular opisthokont Monosiga; the GRKa/4/5/6 family - the canonical of modified predicted amphipathic helix (including unicellular opisthokont Capsaspora), and GRK1/7 family - prenylation motives. However, early GRKs from unicellular oomycetes Phytophthora and Albugo, although loosely grouping with the GRKb/2/3 family, have prenylation motives at the C-terminus, although certain elements of the PH domain can also be detected. The GRK from brown algae Ectocarpus lacks any recognisable C-terminal motive and so does GRK5C from Danio. Species abbreviations are as follows: Acyrthosiphon, <i>Acyrthosiphon pisum</i> (pea aphid); Aedes, <i>Aedes aegypti</i> (yellow fever mosquito); Ailuropoda, <i>Ailuropoda melanoleuca</i> (giant panda); Anopheles, <i>Anopheles gambiae</i> (mosquito); Albugo, <i>Albugo laibachii</i> (oomycete); Apis, <i>Apis mellifera</i> (honey bee); Branchiostoma, <i>Branchiostoma floridae</i> (Florida lancelet; cephalochordate); Bos, <i>Bos taurus</i> (cow); Caenorhabditis, <i>Caenorhabditis elegans</i> (round worm; nematode); Callithrix, <i>Callithrix jacchus</i> (white-tuft-ear marmoset); Canis, <i>Canis lupus familiaris</i> (dog); Caspospora, <i>Caspospora owczazaki</i> (unicellular opisthokont); Ciona, <i>Ciona intestinalis</i> (sea squirt); Cyprinus, <i>Cyprinus carpio</i> (common carp); Drosophila, <i>Drosophila melanogaster</i> (fruit fly; insect); Danio, <i>Danio rerio</i> (zebrafish; teleost fish), Ectocarpus, <i>Ectocarpus siliculosus</i> (brown alga); Enteroctopus, <i>Enteroctopus dofleini</i> (giant octopus); Equus, <i>Equus caballus</i> (horse); Gallus, <i>Gallus gallus</i> (chicken); Homarus, <i>Homarus americanus</i> (American lobster); Homo, <i>Homo</i> sapience (human); Hydra, <i>Hydra magnipapillata</i> (hydra); Monodelphis, <i>Monodelphis domestica</i> (gray short-tailed opossum); Monosiga, <i>Monosiga brevicolis</i> (unicellular opisthokont); Mus, <i>Mus</i> musculus (mouse); Macaca, <i>Macaca mulatta</i> (monkey); Nasonia, <i>Nasonia vitripennis</i> (Jewel wasp); Nematostella, <i>Nematostella vectensis</i> (starlet sea anemone; cnidarian); Ornithorhynchus, <i>Ornithorhynchus anatinus</i> (platypus); Oryctolagus, <i>Oryctolagus cuniculus</i> (rabbit); Oryzias, <i>Oryzias latipes</i> (Japanese medaka fish); Pan, <i>Pan troglodites</i> (chimpanzee); Phytophtora, <i>Phytophtora infestans</i> (oomycete); Rattus, <i>Rattus norvegicus</i> (rat); Schistosoma, <i>Schistosoma mansoni</i> (trematode flatworm); Spermophilus, <i>Spermophilus tridecemlineatus</i> (thirteen-lined ground squirrel; Sus, <i>Sus scrofa</i> (pig); Taeniopygia, <i>Taeniopygia guttata</i> (zebra finch); Trichoplax, <i>Trichoplax adhaerens</i> (placozoan); Tetraodon, <i>Tetraodon nigroviridis</i> (green pufferfish); Tribolium, <i>Tribolium castaneum</i> (beetle); Xenopus, <i>Xenopus laevis</i> (African clawed frog). * - the sequence is truncated at the C terminus.</p

Animals with very low arrestin-1 in the OS show very long time of half recovery.

<p>To calculate the time of half recovery, recovery kinetics were fitted by polynomial nonlinear regression, with R²>0.95, as described in methods. Means +/− SD for four animals per genotype are shown. The data were analyzed by one-way ANOVA with Genotype as main factor followed by Bonferroni post hoc comparison of means. * - p<0.05; ** - p<.001, *** - p<0.001 to WT; + - p<0.05, ++ - p<0.001, +++ - p<0.001 to Arr+/−, a – p<0.005, b – p<0.01, c – p<0.001 to Tr-12^Arr−/−. Phi/rod, photoisomerizations/rod.</p

The rates of photoresponse recovery in mice with different arrestin-1 expression.

<p>The rates of photoresponse recovery in mice with different arrestin-1 expression.</p

Table_3_Effects of cell size and bicarbonate on single photon response variability in retinal rods.pdf

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.</p

Table_1_Effects of cell size and bicarbonate on single photon response variability in retinal rods.pdf

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.</p

Reduced arrestin-1 expression slows down photoresponse recovery.

<p>The intensities of the first (desensitizing) flashes were −0.8, −0.4, 0, or +0.4 <i>log</i>cd*s/m² and second (probe) flash was 0.65 <i>log</i>cd*s/m². The a-wave elicited by the probe flash was plotted as a function of time elapsed after the first flash. Representative recovery curves for indicated genotypes and strengths of desensitizing flash are shown. The interval between the two flashes was varied from 200 to 120,000 ms. The results for desensitizing flash of −0.4 <i>log</i>cd*s/m² were reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022797#pone.0022797-Song1" target="_blank">[10]</a>, and are shown here for comparison. Phi/rod, photoisomerizations/rod. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022797#pone.0022797-Ahmed1" target="_blank">[59]</a></p

Table_2_Effects of cell size and bicarbonate on single photon response variability in retinal rods.pdf

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.</p