Mouse Rod Photoreceptors Research Articles

Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase.

Using 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.

Expression of mouse rod photoreceptor cGMP phosphodiesterase γ subunit in bacteria

We expressed the γ subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGEX-2TK expression vector which produces a cleavable 40 kDa fusion protein. The fusion protein can be isolated in a one step procedure by affinity chromatography on glutathione beads. The yield of purified fusion protein is approximately 10 mg from 1 liter of bacterial culture, or about 3 mg of PDEγ, equivalent to the PDEγ content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDEγ, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDEγ. Immobilized PDEγ binds transducin α subunit charged with GTP, PDE α and β subunits, and, unexpectedly, arrestin (S-antigen).

In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.

Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel.

We have determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel by analysis of cDNA clones and amplified DNA. The open reading frames predicted polypeptides of 690 and 683 residues exhibiting 88% sequence similarity. Sequence comparison indicated that the rod channels consist of a variable 90-residue N-terminal region, a short highly charged segment rich in lysine and glutamate, and a 540-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (59%) of the visual cGMP-gated channel to the olfactory cAMP-gated channel established the existence of a family of cyclic nucleotide-gated ion channel genes. RNA blot analysis revealed transcripts of 3.2 kilobases (kb) in human, mouse, and dog, 3.2, 4.6, and 5.2 kb in bovine, and 3.6 kb in fish. The human channel gene was mapped by polymerase chain reaction of somatic cell hybrid DNAs to chromosome 4 (p14-q13) near the centromere. The mouse channel gene locus (Cncg) was mapped by interspecific backcross haplotype analysis 0.9 centimorgan proximal of the Kit locus on chromosome 5.

Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase α- and β-subunits, and identification of β′-, a putative β-subunit isozyme produced by alternative splicing of the β-subunit gene

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) α- and β-subunits of mouse retinal rod photoreceptors. The open reading frames predict an α-subunit of 100 kDa (856 residues), and a β-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve β-subunit clones predicts the presence in the retina of an additional PDE, termed β, which is generated by alternative splicing of the β-subunit gene, β differs from β only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the α- and β-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of α, β and β.

Synaptic changes in the terminals of rod photoreceptors of albino mice after partial visual cell loss induced by brief exposure to constant light.

Albino mice were exposed to constant light for 7 days and were then transferred to periodic light. After initial photic damage and partial cell loss, the remaining visual cells recovered and survived as a stable population. Regions of the outer nuclear layer containing 4-6 rows of nuclei were more affected than those containing 6-10 rows. Changes in the synaptic structures in the receptor terminals of these two regions were recorded after varying survival periods. Some of the rod terminals had multiple synaptic ribbons and larger numbers of horizontal cell processes and bipolar cell dendrites. The number of terminals with multiple ribbons increased during recovery in periodic light. Morphometry demonstrated that the perimeters of horizontal and bipolar cell processes within the rod terminals were significantly larger than those in age-matched control mice, especially 4 weeks after recovery; they remained significantly larger than controls after 2 and 3 months. We suggest that partial loss of rod cells within a group of cells that are synaptically related to a common bipolar or horizontal cell results in synaptic growth inside the terminals of the surviving cells.