The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

Usually, photoreceptors interact with other retinal cells through the neurotransmitter glutamate. Here we describe a nonsynaptic interaction via a secreted protein, retinoschisin. Using in situ hybridization, we found that from early postnatal life retinoschisin mRNA is present only in the outer retina of the mouse, and with single-cell RT-PCR we demonstrated its localization in rod and cone photoreceptor cells but not in Müller cells. Western blot analyses of proteins from cultured ocular tissues and microdissected outer and inner retinas, as well as from the culture media of these samples, showed that retinoschisin is secreted from the photoreceptor cells. Immunostaining of permeabilized and nonpermeabilized dissociated retinal cells revealed that retinoschisin is mainly inside and outside the photoreceptors, outside bipolar cells, and associated with plasma membranes of Müller cells and inside their distal processes. Because we showed previously that retinoschisin is distributed all over the retina, our current data suggest that after synthesis and secretion by the photoreceptors, retinoschisin reaches the surface of retinal cells and mediates interactions/adhesion between photoreceptor, bipolar, and Müller cells, contributing to the maintenance of the cytoarchitectural integrity of the retina. These interactions may not occur when the gene encoding retinoschisin is mutated, as it occurs in X-linked juvenile retinoschisis, a disease that results in morphological and electrophysiological defects of the retina.

We have generated a mouse with rod photoreceptors overexpressing the ␥ inhibitory subunit (PDE6␥) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6␥ overexpression decreases the rate of rise of the rod response at dim intensities, indicating a reduction in the gain of transduction that may be the result of cytoplasmic PDE6␥ binding to activated transducin ␣ GTP (T ␣ -GTP) before the T ␣ -GTP binds to endogenous PDE6␥. Excess PDE6␥ also produces a marked acceleration in the falling phase of the light response and more rapid recovery of sensitivity and circulating current after prolonged light exposure. These effects are not mediated by accelerating GTP hydrolysis through the GAP (GTPase activating protein) complex, because the decay of the light response is also accelerated in rods that overexpress PDE6␥ but lack RGS9. Our results show that the PDE6␥ binding sites of PDE6 ␣ and ␤ are accessible to excess (presumably cytoplasmic) PDE6␥ in the light, once endogenous PDE6␥ has been displaced from its binding site by T ␣ -GTP. They also suggest that in the presence of T ␣ -GTP, the PDE6␥ remains attached to the rest of the PDE6 molecule, but after conversion of T ␣ -GTP to T ␣ -GDP, the PDE6␥ may dissociate from the PDE6 and exchange with a cytoplasmic pool. This pool may exist even in wild-type rods and may explain the decay of rod photoresponses in the presence of nonhydrolyzable analogs of GTP. Key words: rod; phototransduction; retina; phosphodiesterase; G-protein; RGS protein IntroductionPhotoexcited rhodopsin in a vertebrate rod binds to and activates the G-protein transducin, facilitating the exchange of GTP for GDP on the transducin ␣ subunit (T ␣ or GNAT1). The T ␣ -GTP then binds to the inhibitory ␥ subunit (PDE6␥) of the phosphodiesterase effector enzyme (PDE6), relieving the inhibition of the PDE6 ␣ and ␤ catalytic subunits. Activated PDE6 hydrolyzes cGMP, leading to the closing of the cGMP-gated channels in the outer segment. This produces the hyperpolarizing light response that signals the detection of the light to the rest of the nervous system .The turnoff of the photoreceptor response and reopening of the channels requires the inactivation of rhodopsin by phosphorylation and subsequent binding of arrestin, as well as the return of the PDE6 to its dark resting level by hydrolysis of T ␣ -GTP back to T ␣ -GDP. The intrinsic rate of transducin GTP hydrolysis is slow but is facilitated by interaction of transducin with other proteins . The first of these to be identified was PDE6␥, which was initially thought to act by itself to accelerate GTP hydrolysis (Arshavsky and Bownds, 1992) but was later shown to have no effect on the rate of hydrolysis in isolation (Angleson and Wensel, 1993;Antonny et al., 1993) and to require additional components, subsequently identified as RGS9 -1 (He et al., 1998), G␤5 (Makino et al., 1999, and a membrane anchor protein, R9AP (Hu and Wensel, 2002). These together form a GTPase activating protein (GAP) complex that is essential for the rapid conve...

Mutations in the β-subunit of cGMP-phosphodiesterase (PDE6β) can lead to either progressive retinal disease, such as human retinitis pigmentosa (RP), or stationary disease, such as congenital stationary night blindness (CSNB). Individuals with CSNB in the Rambusch pedigree were found to carry the H258N allele of PDE6B (MIM# 180072); a similar mutation was not found in RP patients. This report describes an individual carrying the H258N allele, who presented with generalized retinal dysfunction affecting the rod system and a locus of dysfunction at the rod-bipolar interface. Also described are preclinical studies in which transgenic mice with the H258N allele were generated to study the pathophysiological mechanisms of CSNB. While Pde6b rd1 /Pde6b rd1 mice have severe photoreceptor degeneration, as in human RP, the H258N transgene rescued these cells. The cGMP-PDE6 activity of dark-adapted H258N mice showed an approximate three-fold increase in the rate of retinal cGMP hydrolysis: from 130.1 nmol × min −1 × nmol −1 rhodopsin in wild-type controls to 319.2 nmol × min −1 × nmol −1 rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6β activity by the regulatory PDE6γ subunit is blocked by this mutation. In the albino (B6CBA × FVB) F2 hybrid background, electroretinograms (ERG) from H258N mice were similar to those obtained from affected Rambusch family members, as well as humans with the most common form of CSNB (X-linked), demonstrating a selective loss of the b-wave with relatively normal awaves. When the H258N allele was introduced into the DBA background, there was no evidence of selective reduction in b-wave amplitudes; rather a-and b-wave amplitudes were both reduced. Thus, factors other than the PDE6B mutation itself could contribute to the variance of an

The bovine a and murine P subunits of rod-photoreceptor cGMP-phosphodiesterase (PDEL and PDEp) were expressed in adenovirus-transformed 293 human embryonic kidney cells. RNA blots from transfected cells showed transcripts of 3.0 and 2.8 kb corresponding to PDEL and PDEp, respectively. Protein expression was analyzed by using affnity-purified antibodies against cGMP-PDE on immunoblots and by immunoprecipitation. PDEa and PDEp exhibited the expected mobility (and thus apparent molecular size) and had cGMP hydrolytic activity. Reconstitution of the PDE a13 heterodimer with the expressed proteins increased by =6-fold the activity of the individual a and (8 subunits. Addition of expressed (8 subunit to retinal extracts from 9-to 10-day-old rd/rd mice (which have only normal a and Y subunits of rod cGMP-PDE and thus minimal activity) increased enzyme activity by -3-fold. Our results therefore demonstrate that photoreceptor-specific cGMP-PDE can be synthesized in human kidney cells with consequent expression of enzymatic activity.Rod-photoreceptor cGMP-phosphodiesterase (cGMP-PDE) is one of the key enzymes of the visual phototransduction cascade in the vertebrate retina (1). The holoenzyme is a heterotetrameric complex, consisting of two large catalytic subunits, a (88 kDa; PDEa) and P (84 kDa; PDE9), and two identical inhibitory subunits y (11 kDa) (2, 3). cGMP-PDE activity is stimulated in vivo by removal of the inhibitory y subunits by activated transducin (4). cGMP-PDE can be stimulated in vitro by incubation with histones (5) or by limited proteolytic digestion with trypsin (6).The cDNAs encoding the three subunits of cGMP-PDE from different animal species have been cloned, and their nucleotide and amino acid sequences have been determined (7)(8)(9)(10). Sequence homology between the a and 8 subunits from bovine (9) and murine (11) rod photoreceptors is -72% and 74%, respectively. Each of these subunits contains two putative noncatalytic cGMP-binding sites, two y subunitbinding sites, a putative catalytic domain, and the CAAX motif for isoprenylation (9,(12)(13)(14)(15). In rod photoreceptor cells the a and ,B subunits are farnesylated and geranylgeranylated, respectively, which facilitates their association with the disc membranes. The domain organization of these proteins suggests that they can have independent cGMP-hydrolytic activity.To carry out studies on the structure and function of cGMP-PDE in normal retinas and in those affected with genetic diseases, each of the biologically active subunits of the enzyme is necessary. Although the separation of the catalytic subunits is possible under denaturing conditions, no reports have been published to date on the isolation of active PDEa or PDE,9 using classical preparative biochemical procedures. Difficulties inherent to these methods can be overcome with the use of recombinant DNA technology, which has been applied in many instances to express large amounts of active proteins in bacterial or mammalian systems. However, previous attempts to express the ...