Rod photoreceptor cGMP-phosphodiesterase: analysis of alpha and beta subunits expressed in human kidney cells.

The catalytic core of photoreceptor-specific cGMPphosphodiesterase (PDE) consists of two subunits, PDE␣ and PDE␤, that are homologous and have similar domain organization but are encoded by different genes. We have examined the PDE␣ and PDE␤ mRNA steady-state and protein levels as well as the biosynthesis rate of these proteins in developing and fully differentiated retinas. We have also determined the translational efficiency of PDE subunits and the role of their mRNA structures in regulating protein synthesis. In mature retinas, PDE␣ and PDE␤ are represented by ϳ1.5 ؋ 10 8 and 7.5 ؋ 10 8 copies/g retinal mRNA, respectively. The levels of these transcripts in developing photoreceptors (P10) are approximately 75% of those at P30. Quantification of protein concentration indicated that PDE␣ and PDE␤ are equally expressed in developing and fully differentiated photoreceptors. Furthermore, the PDE␣/PDE␤ ratios obtained throughout a 2-h pulsechase period revealed a similar turnover rate for both subunits. The observed discordance between the mRNA and protein levels of PDE␣ and PDE␤ suggested posttranscriptional regulation of their expression. We found that PDE␣ mRNA is translated more efficiently than either of the two PDE␤ transcripts expressed in retina. Therefore, the lower level of PDE␣ mRNA is compensated by its more efficient translation to achieve equimolar expression with PDE␤. We also analyzed the effect of PDE␣ and PDE␤ mRNA 5-and 3-untranslated regions as well as that of their coding regions on protein synthesis. We determined that the PDE-coding regions play a critical role in the differential translation of these subunits.Signal transduction in the vertebrate rod and cone photoreceptors is mediated by a cascade of protein interactions/activations that lead to hydrolysis of cGMP by a photoreceptor-specific cGMP-phosphodiesterase (PDE) 1 followed by closure of the cGMP-gated channels on the plasma membrane (1). The catalytic core of cone PDE is composed of two identical ␣Ј subunits, whereas that of the rod enzyme is a heterodimer (PDE␣, 88 kDa and PDE␤, 84 kDa) (2, 3). Although each of these three proteins is encoded by a different gene, they share significant homology in both amino acid and nucleotide sequences. Cone and rod PDEs have similar domain organization and contain two cGMP-binding site motifs, a catalytic site and a CAAX box signal for post-translational modification (4). Why the rod holoenzyme consists of two different subunits is still unknown. Based on classical biochemical studies carried out with the bovine rod cGMP-PDE, it has been accepted that PDE␣ and PDE␤ are present in a 1:1 ratio and that they are assembled in a heterodimeric catalytic core (2, 3, 5). This coordinated expression of PDE␣ and PDE␤ in rod photoreceptors must be regulated at the transcriptional and/or post-transcriptional level so that a final equimolar ratio of these proteins is achieved. However, there is some indirect evidence suggesting the possibility that the rod PDE catalytic core is not only present as a heterodi...

mentioning

confidence: 84%

Differential Expression of Rod Photoreceptor cGMP-Phosphodiesterase α and β Subunits

Piri

Yamashita

Shih

et al. 2003

“…To investigate whether Crx is able to directly transactivate the ␤-PDE promoter, we transiently overexpressed Crx in 293 human embryonic kidney cells in cotransfections with different deletion mutants of the ␤-PDE promoter. These cells do not endogenously produce rod-specific phosphodiesterases includ-ing ␤-PDE and have been used previously for transient transfections to study transcriptional regulation of the ␤-PDE gene (14,15,20,27). Fig.…”

Section: Resultsmentioning

confidence: 99%

The Rod cGMP-phosphodiesterase β-Subunit Promoter Is a Specific Target for Sp4 and Is Not Activated by Other Sp Proteins or CRX

Lerner

Gribanova²,

Whitaker

et al. 2002

The ␤-subunit of cGMP-phosphodiesterase (␤-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. ␤-PDE promoter deletions showed that the ؊45/؊23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical ␤-PDE transcriptional regulator in vivo. Interestingly, although the ␤/GC element (؊59/؊49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced ␤-PDE promoter activity. Thus, the Sp4-mediated differential activation of the ␤-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the ␤-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich ␤/TA sequence located within the ؊45/؊23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.One of the key components of the phototransduction cascade that takes place in rod photoreceptors is the heterotetrameric (␣␤␥ 2 ) cGMP-phosphodiesterase (1). The gene encoding the ␤-subunit of the human enzyme (␤-PDE) 1 has been well characterized and consists of 22 exons encompassing ϳ43 kb of genomic DNA (2). Genetic defects in this gene have been linked to retinal degeneration in several animal species and human (3-9). There is increasing evidence that abnormalities in transcriptional regulatory components of different genes contribute significantly to or directly cause pathological phenotypes in the retina (10 -13). Therefore, further studies on the transcriptional regulation of rod-specific ␤-PDE gene will identify additional genes important for retinal function and structural integrity and will ultimately help to establish the molecular mechanisms crucial for retina-specific expression of this and perhaps some other genes.We recently reported our initial results on the transcriptional control mechanisms that take place in the human ␤-PDE 5Ј-flanking region (14). Mutational analysis of the ␤-PDE promoter tested both in vitro and ex vivo, and confirmed by the generation of transgenic Xenopus expressing mutant ␤-PDE promoter/green fluorescent protein fusion constructs in vivo, revealed a minimal promoter region, from Ϫ93 to ϩ53, that supports high levels of rod-specific transcription (14). Two enhancer elements were localized within this minimal promoter, ␤Ap1/NRE and ␤/GC, that interact with nuclear factors and activate transcription from the ␤-PDE promoter.To continue the systematic analysis of the ␤-PDE promoter...

“…Yet, PDE6, the key enzyme of vision, remains arguably one of the most obscure G protein effectors in terms of understanding its structure/function relationship. Difficulties in the development of an efficient expression system for PDE6 have precluded the systematic mutational analysis of the enzyme (17)(18)(19). Our attempts to express functionally wild-type PDE6␣Ј and co-express PDE6␣Ј with P␥ using the Baculovirus/Sf9, COS7, or retinoblastoma Y79 cell systems have also been unsuccessful.…”

Section: Discussionmentioning

confidence: 99%

“…Progress in the investigation of the structure/function of PDE6 and the mechanism of PDE6 inhibition by P␥ has been slowed by the lack of an efficient expression system for PDE6 (17,18). Our approach to developing a system for PDE6 expression and mutagenesis included the construction of chimeras between PDE6␣Ј and cGMP-binding, cGMP-specific PDE (PDE5 family) (19).…”

mentioning

confidence: 99%

Identification of the γ Subunit-interacting Residues on Photoreceptor cGMP Phosphodiesterase, PDE6α′

Granovsky

Artemyev

2000

Photoreceptor cGMP phosphodiesterases (PDE6 1 family) function as effector proteins in the vertebrate visual transduction, which is mediated by the rhodopsin-coupled G protein, transducin (1-3). Retinal rod PDE6 is composed of two catalytic PDE6␣␤ subunits each tightly associated with the smaller inhibitory ␥ subunit (P␥) (4 -6). Cone PDE consists of two identical PDE␣Ј subunits complexed with two copies of the cone-specific P␥ subunit (7-9). The catalytic subunits of rod and cone PDE, as well as the respective P␥ subunits, share a high degree of homology (9 -10). The key role of P␥ is to inhibit cGMP hydrolysis by the catalytic subunits in the dark. Upon light stimulation of photoreceptors, PDE6 is activated by GTPbound transducin-␣, which displaces P␥ from the enzyme catalytic core.Two regions of P␥ are principally involved in the interaction with the PDE6 catalytic subunits, the central polycationic region (residues 24 -45 of rod P␥) and the P␥ C terminus. The C terminus of P␥ constitutes the key inhibitory domain, whereas the polycationic region enhances the overall affinity of P␥ toward PDE6 catalytic subunits (11)(12)(13)(14). A cross-linking study localized the P␥ C-terminal binding site on PDE6␣ to residues 751-763 (residues 749 -761 of PDE6␤ or PDE6␣Ј) within the broader PDE6 catalytic domain (15). Our further analysis of the interaction between fluorescently labeled P␥ and PDE6␣␤ suggests that the C terminus of P␥ inhibits PDE6 activity by physically blocking the PDE catalytic site (16).Progress in the investigation of the structure/function of PDE6 and the mechanism of PDE6 inhibition by P␥ has been slowed by the lack of an efficient expression system for PDE6 (17, 18). Our approach to developing a system for PDE6 expression and mutagenesis included the construction of chimeras between PDE6␣Ј and cGMP-binding, cGMP-specific PDE (PDE5 family) (19). PDE5 and PDE6 share a common domain organization, i.e. two noncatalytic cGMP-binding sites located N-terminally to the conserved PDE catalytic domain (20). Furthermore, PDE5 and PDE6 display a high homology (45-48% identity) between catalytic domains, a strong substrate preference for cGMP, and similar patterns of inhibition by competitive inhibitors such as zaprinast, dipyridamole, and sildenafil (20 -23). Unlike PDE6, PDE5 is readily expressed using the baculovirus/insect cell system (24, 25). Earlier, we reported (19) the functional expression and characterization of a chimeric PDE6␣Ј/PDE5 enzyme containing the PDE6␣Ј noncatalytic cGMP-binding sites and the PDE5 catalytic domain. In this study, we generated chimeric PDE6␣Ј/PDE5 enzymes that contain the P␥ C-terminal binding site and that are potently inhibited by P␥. Ala-scanning mutational analysis of the P␥-binding site, using chimeric PDE as a template, revealed the key interaction residues and provided structural justification for the mechanism of PDE6 inhibition.