Light-dependent redistribution of visual arrestins and transducin subunits in mice with defective phototransduction

Journal ArticlePURPOSE: The light-dependent redistribution of phototransduction components in photoreceptor cells plays a role in light adaptation. Upon illumination, rod and cone arrestins (Arr and cArr) translocate from the inner to the outer segments while transducin subunits (Talpha, Tbetagamma) translocate in the opposite direction. The underlying translocation mechanisms are unclear. This study examines these previously demonstrated translocation in mice with defective phototransduction. METHODS: The distribution of Arr, cArr, Talpha, and Tbetagamma was examined using immunoblotting and immunocytochemistry in dark- and light-adapted single knockout mice lacking G-protein coupled receptor kinase 1 (Grk1-/-) and double knockout mice lacking GRK1 and transducin alpha subunit (Grk1-/-/Gnat1-/-), or lacking GRK1 and arrestin (Grk1-/-/Arr-/-). RESULTS: Arr redistributed in the light to the outer segments in Grk1-/- mice as well as in Grk1-/-/Gnat1-/- double knockout retinas. Immunoblotting revealed that approximately 25-50% of Arr associated with the membrane in light-adapted wild-type, Grk1-/- and Gnat1-/-/Grk1-/- mouse retinas. In contrast, cArr did not stably associate with light-adapted membranes in either wild-type or Grk1-/- retinas under our experimental conditions, but redistributed to the cone outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurred in both wild-type and Grk1-/-/Arr-/- double knockout photoreceptors. However, Tbetagamma subunits did not redistribute in the absence of Talpha, suggesting that transducin only translocates as an intact heterotrimer. CONCLUSIONS: We conclude that in rods, Arr redistribution requires neither rhodopsin phosphorylation nor phototransduction, suggesting the presence of another light-dependent pathway to trigger translocation. In cones, the light-dependent movement of cArr appears to be independent of stable association with the cone pigments. The light-dependent translocations of Arr and transducin subunits in opposite directions appear to be based on independent mechanisms

Expression and characterization of human PDEδ and its Caenorhabditis elegans ortholog CEδ

AbstractCyclic GMP phosphodiesterase (PDE) is rod photoreceptor disk membrane-associated via C-terminal lipid tails. PDEδ, a recently identified subunit, was shown to disrupt PDE/membrane interaction under physiological conditions, without affecting PDE catalytic activity. We found that a PDEδ ortholog from the eyeless nematode Caenorhabditis elegans (termed CEδ) solubilizes bovine PDE in vitro with an EC50 very similar to PDEδ. Immobilized PDEδ and CEδ both bind, in addition to bovine PDE, an N-terminal fragment of human retinitis pigmentosa GTPase regulator, but not rhodopsin kinase and Ran binding protein 1. The results suggest that PDEδ and CEδ may regulate membrane binding of a variety of proteins in photoreceptors and other tissues

Cloning and molecular characterization of cGMP-gated ion channels from rod and cone photoreceptors of striped bass ( M. saxatilis ) retina

Journal ArticleVertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel alpha subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both alpha (CNGA3) and beta (CNGB3) channel subunits. For alpha, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For beta, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca(2+)-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species

Calcium-sensitive particulate guanylyl cyclase as a modulator of cAMP in olfactory receptor neurons

Journal ArticleThe second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events

Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase

Journal ArticleUsing recombinant baculovirus vectors, the three subunits of mouse rod photoreceptor cGMP phosphodiesterase (PDE) (alpha beta gamma 2) have been expressed in insect cells. The recombinant alpha,beta subunits accumulate to 5 mg/liter culture, but most (98%) of the expressed polypeptides are insoluble. In the soluble fraction, individually expressed alpha and beta subunits showed insignificant PDE activity, but coexpression (by coinfection) of alpha beta subunits elevated PDE activity 7-fold and coexpression of alpha beta gamma up to 15-fold. The soluble expressed holoenzyme associated with ROS membranes under isotonic, but not hypotonic, conditions. The Km of the soluble holoenzyme was 11-16 microM both for coexpressed alpha beta subunits and for alpha beta gamma subunits, similar to the Km (6-80 microM) of native PDE. Site-directed mutagenesis of cysteine to serine in the C-terminal CAAX box of both alpha and beta subunits substantially decreased the protein expression level, abolished post-translational isoprenylation, and prevented subunit binding to the rod outer segment (ROS) membranes. The mutant holoenzyme, however, showed a cGMP hydrolytic activity comparable with that of the normal recombinant enzyme. These results suggest that both alpha and beta subunits are required for the formation of a functional enzyme and that isoprenylation of the subunits is essential for membrane association and stability of PDE

Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A)

Journal ArticlePURPOSE: To describe the clinical features and genetic analysis of a family with an autosomal dominant cone dystrophy (adCD). METHODS: Selected members of a family with an autosomal dominant cone dystrophy underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and genomic fragments were amplified by PCR. Linkage to locus D6S1017 was established. DHPLC mutational analysis and direct sequencing were used to identify a mutation in GUCA1A, the gene encoding the guanylate cyclase activating protein 1 (GCAP1). RESULTS: Of 24 individuals who are at risk of the disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinogram performed on one proband revealed profoundly subnormal and prolonged photopic and flicker responses, but preserved scotopic ERGs, consistent with a cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a novel L151F mutation in GCAP1. Like the E155G mutation, this mutation occurs in the EF4 hand domain, a region of GCAP1 critical in conferring calcium sensitivity to the protein. The leucine at this position is highly conserved among vertebrate guanylate cyclase activating proteins. CONCLUSIONS: A novel L151F missense mutation in the EF4 high affinity Ca2+ binding site of GCAP1 is linked to adCD in a large pedigree. The cone dystrophy in this family shares clinical and electrophysiologic characteristics with other previously described adCD caused by mutations in GUCA1A

Novel GCAP1 missense mutation (L151F) in a large family with autosomal dominant cone-rod dystrophy (adCORD)

Journal ArticlePURPOSE: To elucidate the phenotypic and biochemical characteristics of a novel mutation associated with autosomal dominant cone-rod dystrophy (adCORD). METHODS: Twenty-three family members of a CORD pedigree underwent clinical examinations, including visual acuity tests, standardized full-field ERG, and fundus photography. Genomic DNA was screened for mutations in GCAP1 exons using DNA sequencing and single-strand conformational polymorphism (SSCP) analysis. Function and stability of recombinant GCAP1-L151F were tested as a function of [Ca(2+)], and its structure was probed by molecular dynamics. RESULTS: Affected family members experienced dyschromatopsia, hemeralopia, and reduced visual acuity by the second to third decade of life. Electrophysiology revealed a nonrecordable photopic response with later attenuation of the scotopic response. Affected family members harbored a C-->T transition in exon 4 of the GCAP1 gene, resulting in an L151F missense mutation affecting the EF hand motif 4 (EF4). This change was absent in 11 unaffected family members and in 100 unrelated normal subjects. GCAP1-L151F stimulation of photoreceptor guanylate cyclase was not completely inhibited at high physiological [Ca(2+)], consistent with a lowered affinity for Ca(2+)-binding to EF4. CONCLUSIONS: A novel L151F mutation in the EF4 hand domain of GCAP1 is associated with adCORD. The clinical phenotype is characterized by early cone dysfunction and a progressive loss of rod function. The biochemical phenotype is best described as persistent stimulation of photoreceptor guanylate cyclase, representing a gain of function of mutant GCAP1. Although a conservative substitution, molecular dynamics suggests a significant change in Ca(2+)-binding to EF4 and EF2 and changes in the shape of L151F-GCAP1

Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2

Journal ArticlePURPOSE: To identify gene arrangement, chromosomal localization, and expression pattern of mouse guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and photoreceptor guanylate cyclase activators. METHODS: The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry. RESULTS: In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA transcripts, and immunolocalization of the gene products were fully characterized. The GCAP tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones. GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2 staining within the outer segment and outer plexiform layers. CONCLUSIONS: Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse and human suggest an ancient gene duplication-inversion event preceding mammalian diversification. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest roles of these Ca-binding proteins in addition to regulation of phototransduction

Molecular characterization of human and mouse photoreceptor guanylate cyclase-activating protein (GCAP) and chromosomal localization of the human gene

Journal ArticleGuanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1)

Identification and light-dependent translocation of a cone-specific antigen, (Cone Arrestin) recognized by monoclonal antibody 7G6

Journal ArticlePURPOSE: To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker. METHODS: 7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen. RESULTS: Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin. CONCLUSIONS: Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies