Isolation and characterization of a cDNA encoding the α′ subunit of human cone cGMP-phosphodiesterase

doi:10.1016/0378-1119(95)00585-4

Gene

Volume 166, Issue 2, 12 December 1995, Pages 205-211

https://doi.org/10.1016/0378-1119(95)00585-4 Get rights and content

Abstract

A human cDNA (α′-PDE) encoding the α′ catalytic subunit of cone photoreceptor cGMP-phosphodiesterase has been isolated and characterized. The nucleotide sequence of 2980 bp contains an open reading frame encoding an 859-aminoacid (aa) protein with a calculated molecular mass of 99 kDa. Northern blot analysis of human retinal mRNA hybridized with the α′-PDE cDNA revealed a signal corresponding to a 3.2-kb transcript. Comparison of the deduced aa sequence of human cone α′-PDE with corresponding proteins isolated from bovine and chicken retinas shows 89 and 83% identity, respectively, and indicates that α′-PDE has been very well conserved in the evolutionary process. Human cone α′-PDE is highly homologous to rod α-PDE and β-PDE, suggesting that these proteins may have a close phylogenetic relationship.

References (38)

W. Baehr et al.
Isolation and characterization of cGMP phosphodiesterase from bovine rod outer segments
J. Biol. Chem.
(1979)
D.B. Farber et al.
The β subunit of cyclic GMP phosphodiesterase mRNA is deficient in canine rod-cone dysplasia 1
Neuron
(1992)
P.G. Gillespie et al.
Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography
J. Biol. Chem.
(1988)
R.L. Hurwitz et al.
cGMP phosphodiesterase in rod and cone outer segments of the retina
J. Biol. Chem.
(1985)
R.J. Jackson
Cytoplasmic regulation of mRNA function: The importance of the 3′ untranslated region
Cell
(1993)
F. Jurnak
The three-dimensional structure of c-H-ras p21: implications for oncogene and G protein studies
Trends Biochem. Sci.
(1988)
V.M. Lipkin et al.
β-Subunit of bovine rod photoreceptor cGMP phosphodiesterase
J. Biol. Chem.
(1990)
S.L. Moores et al.
Sequence dependence of protein isoprenylation
J. Biol. Chem.
(1991)
B. Oppert et al.
Identification of the retinal cyclic GMP phosphodiesterase inhibitory γ-subunit interaction sites on the catalytic α-subunit
J. Biol. Chem.
(1991)
B. Oppert et al.
Identification of the γ-subunit interaction sites in the retinal cyclic-GMP phosphodiesterase β-subunit
Biochem. Biophys. Res. Commun.
(1991)

Y.A. Ovchinnikov et al.

Cyclic GMP phosphodiesterase from bovine retina: amino acid sequence of the α-subunit and nucleotide sequence of the corresponding cDNA

FEBS Lett.

(1987)

S.J. Pittler et al.

Molecular characterization of human and bovine rod photoreceptor cGMP phosphodiesterase α-subunit and chromosomal localization of the human gene

Genomics

(1990)

A. Rashtchian et al.

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: Application to genomic and cDNA cloning

Anal. Biochem.

(1992)

S.D. Stroop et al.

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

J. Biol. Chem.

(1989)

F.M. Ausubel et al.

J.K. Bentley et al.

Regulation and function of cyclic nucleotides

Curr. Opin. Cell Biol.

(1992)

C. Bowes et al.

Retinal degeneration in the rd mouse is caused by a defect in the β subunit of rod cGMP-phosphodiesterase

Nature

(1990)

C. Bowes et al.

Localization of a retroviral element within the rd gene coding for the β subunit of cGMP phosphodiesterase

H. Charbonneau et al.

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases

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Citation Excerpt :
Upon light stimulation of photoreceptors, PDE6s are stimulated through removal of these small inhibitory subunits by activated GTP-bound transducin [12]. PDE6α, PDE6β and PDE6α′ are encoded by different genes, and their cDNAs have been cloned in human and many other species [13–18]. Comparison of the deduced amino acid sequences reveals that the homology among PDE6α, PDE6β and PDE6α′ is as high as about 62–72%, and that the PDE6 sequences and subunit structure are well-conserved among different species [1,19].
Human photoreceptor cGMP-phosphodiesterases (PDE6s) are important reagents in PDE inhibitor discovery. However, recombinant human PDE6s have not been expressed, and isolation of native human PDE6s is highly difficult. In this study, the catalytic subunit(s) of human rod and cone PDE6s (PDE6αβ and PDE6α′, respectively) were co-expressed or expressed separately as catalytically active enzymes. Sildenafil inhibited both the recombinant PDE6s in a dose-dependent manner with K_i values of 94 and 98 nM, respectively. These K_i values were four-fold higher than that (25 nM) of a human native PDE6 preparation. Similarly, 3-isobutyl-1-methylxanthine (IBMX)’s K_i values for the recombinant PDE6s were five- to eight-fold higher than that of the native enzyme. However, E4021 and zaprinast exhibited much (30–80-fold) lower potencies for the recombinant PDE6s than for the native enzyme. Additional PDE5 inhibitors representing other structural classes and possessing different selectivity against native PDE6 also showed different potencies against the recombinant and native PDE6s. In particular, one class of xanthine analogues exhibited significantly (5–15-fold) higher potencies for the recombinant PDE6s than for the native enzyme. Our data demonstrates that the recombinant and native PDE6s exhibit differential sensitivity to inhibitors, and cautions the use of recombinant catalytic subunits of PDE6 in drug discovery or in structural/functional studies.
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To identify novel retina-specific genes systematically, we are performing expression profiling of retina ESTs that have been assembled in the human UniGene clusters. In this study, we report the 2619-bp full-length cDNA cloning and genomic organization of a gene corresponding to an EST cluster that was demonstrated to be exclusively present in retinal tissue. Alignment of the deduced amino acid sequence to sequence from protein databases revealed this gene, termed MPP4, to be a member of the membrane-associated guanylate kinase (MAGUK) protein family. It consists of 637 amino acids and contains the characteristic MAGUK motifs: an N-terminal PDZ domain, a central src homology 3 region (SH3), and a C-terminal guanylate kinase-like (GUK) domain. Due to the presence of only one PDZ motif, MPP4 is part of the p55 subfamily, named after the major palmitoylated erythrocyte membrane protein p55/MPP1. MAGUK proteins serve as molecular scaffolds to coordinate the membrane-associated cytoskeleton, ion channel and receptor clustering, signaling pathways, and the formation of cellular junctions. The abundant expression of MPP4 in the human retina suggests an important but so far unknown function in this tissue. Colocalization of MPP4 and autosomal recessive retinitis pigmentosa 26 (RP26) on chromosome 2q31–q33 makes this transcript an attractive candidate for the disease gene.
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Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein γ subunit (G_γc, previously named G_γ8), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Onget al.,1995,J. Biol. Chem.270: 8495–8500). We report here the characterization of human G_γcand its gene structure. Human G_γcsubunit shares a high degree of sequence identity with the corresponding bovine G_γcisoform (85%) and human rod G_γ1(63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-G_γcantibodies. Nucleotide sequence analysis of the G_γcgene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod G_γ1gene (GNGT1). By using fluorescencein situhybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the G_γcgene structure would facilitate the identification of genetic defects associated with cone degeneration.
Cloning of the cDNA encoding rod photoreceptor cGMP-phosphodiesterase α and γ subunits from the retinal degenerate Labrador Retriever dog
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The nucleotide (nt) sequence of the Cdna encoding the retinal rod cyclic 3′5′-gmp phosphodiesterase (PDE) α and γ subunits from two strains of dogs - (i) Labrador Retrievers homozygous for autosomally recessively inherited rod-cone degeneration and (ii) the wild-type Beagle - are reported. Cloning of these subunits was accomplished by polymerase chain reaction using retinal CDNA libraries as templates. The nt sequence of α pde predicts a 861-amino-acid polypeptide which is 97·7 per cent and 96·9 per cent identical to the bovine and human counterparts, respectively. pde γ encodes an 87-amino-acid polypeptide differing from bovine and murine gamma subunits by only one amino acid. Since no differences were found between these two strains of dogs, the cause of the Labrador Retriever's degeneration remains to be determined.
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Isolation and characterization of a cDNA encoding the α′ subunit of human cone cGMP-phosphodiesterase

Abstract

J. Biol. Chem.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Cell

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Genomics

Anal. Biochem.

J. Biol. Chem.

Regulation and function of cyclic nucleotides

Curr. Opin. Cell Biol.

Retinal degeneration in the rd mouse is caused by a defect in the β subunit of rod cGMP-phosphodiesterase

Nature

Localization of a retroviral element within the rd gene coding for the β subunit of cGMP phosphodiesterase

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases