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

Abstract

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...

Functional Analysis of the Rod Photoreceptor cGMP Phosphodiesterase α-Subunit Gene Promoter

“…In this paper, we show that the PDE6A promoter is contained within a 100-bp segment of the upstream region and requires Nrl and Crx for full transcriptional activity. Our data and that from studies of the PDE6B gene promoter (7), which shows greater activity but contains essentially the same binding sites, indicate that promoter strength is at least in part due to the specific sequence and the combinatorial array of cis-elements within a given promoter.…”

“…Both core promoters are contained within 105 bp upstream of the transcription start point, both initiate transcription at one major site and one minor site, and both consist of a similar array of cis-elements including a GC box, an E-box, and Crx and Nrl binding sites, yet there is a 5-8-fold lower activity of the Pde6a promoter (7,11). The primary differences are the spatial arrangement of the ciselements, the sequence of the NRE/AP1 site, and the absence of repetitive elements upstream of the Pde6b promoter.…”

“…Dissection of promoter architecture may also be important to develop safe gene therapy vectors with controlled targeted expression in specific cell types. Our initial characterization of the PDE6 ␣-subunit gene promoter indicated that it shows very weak activity compared with other photoreceptor gene promoters including the PDE6B gene promoter (7). We suggest that the relatively weak expression of this promoter could be useful where overexpression with the well-characterized, most commonly used rhodopsin promoter could induce, rather than reverse, retinal degeneration (8,9).…”

Rod phosphodiesterase-6 PDE6A and PDE6B Subunits Are Enzymatically Equivalent

“…These conserved differences apparently include determinants for homodimerization of PDE6C and heterodimerization of PDE6AB. Heterodimerization of rod PDE6 is a potential mechanism to control enzyme expression, folding, and assembly via a rate-limiting translation of one of the catalytic subunits (38). In addition, rod and cone PDE6 may be specialized for selective interactions with regulatory proteins.…”

The Inhibitory γ Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure

“…In stark contrast, however, only limited knowledge has been gained about the P␥-interacting sites on P␣␤ because this analysis is particularly difficult. Heterologous expression and mutagenesis of the ␣␤ catalytic heterodimer with high activity has not been successful thus far (26 -28), probably due to complex regulation of expression and assembly of the ␣␤ subunits (12,29,30). For this reason, PDE6 activity assays using native P␣␤ have been a popular approach for the study of P␥/P␣␤ interactions (4,6,10,24,25,31,32).…”