Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65؊/؊ mutant mice, which do not produce the chromophore 11-cisretinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.

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The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.

G protein-coupled receptors (GPCRs) are ubiquitous and essential in modulating virtually all physiological processes. These receptors share a similar structural design consisting of the seven-transmembrane alpha-helical segments. The active conformations of the receptors are stabilized by an agonist and couple to structurally highly conserved heterotrimeric G proteins. One of the most important unanswered questions is how GPCRs couple to their cognate G proteins. Phototransduction represents an excellent model system for understanding G protein signaling, owing to the high expression of rhodopsin in rod photoreceptors and the multidisciplinary experimental approaches used to study this GPCR. Here, we describe how a G protein (transducin) docks on to an oligomeric GPCR (rhodopsin), revealing structural details of this critical interface in the signal transduction process. This conceptual model takes into account recent structural information on the receptor and G protein, as well as oligomeric states of GPCRs.

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH؊/؊) mouse. Histological analysis and electron microscopy of retinas from 6-to 8-week-old prRDH؊/؊ mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.Reduction and oxidation of retinoids are key reactions of the retinoid cycle (visual cycle), which is critical for the production of the chromophore of rhodopsin, 11-cis-retinal (1, 2). When light strikes the visual pigments (rhodopsin and cone opsins) in photoreceptors, it causes the 11-cis-retinylidene chromophore to isomerize to its all-trans configuration, before all-trans-retinal is released from the binding site of the pigments (3) (see Scheme 1). The NADPH-dependent reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigment. The reduction occurs directly on the cytoplasmic surface of outer segment disk membranes. Once all-trans-retinal escapes into the internal disk space, it is pumped out to the cytosol by a photoreceptorspecific ATP-binding transporter (4 -8). Several all-trans-retinol dehydrogenases (RDHs) 1 from the photoreceptor cells have been identified. First, Haeseleer et al. (9) cloned a cone-specific enzyme from the short-chain dehydrogenase/reductase (SDR) family with properties that suggest participation in the retinoid cycle. Next, Rattner and colleagues (10) reported the identification of a novel member of the SDR family, photoreceptor RDH (prRDH or RDH8), that localized to photoreceptors and possessed enzymatic properties closely matching those previously reported for RDH activity in ROS. The authors suggested that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment. The sequence homology among SDRs is typically low (20 -40%), but the structural homology is high and most protein folds are conserved (11). prR...

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho؊/؊ mice. ROS of mice heterozygous for the Rho gene deletion (Rho؉/؊) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho؉/؊ mice as compared with age-matched WT mice, the length of ROS was shorter by 30 -40%, and the average diameter of ROS was reduced by ϳ20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was ϳ60% in Rho؉/؊ mice. Rho content in the eyes was reduced by ϳ43% and 11-cis-retinal content in the eye was reduced by ϳ38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho؉/؊ mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at ϳ4.5 nm ؊1 , indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho؉/؊ disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho؉/؊ mice are due to structural changes of the whole ROS and not due to a lower density of Rho.

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas‐Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease‐causing mutation in approximately one‐third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone‐rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone‐rod homeobox gene, CRX. Based on this large survey, the prevalence of disease‐causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation‐specific studies. Hum Mutat 17:42–51, 2001. © 2001 Wiley‐Liss, Inc.

Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) were given a single intravitreous injection of an E1-, partial E3-, E4-deleted adenoviral vector expressing human pigment epithelium- derived factor (AdPEDF.11). Doses ranging from 10(6) to 10(9.5) particle units (PU) were investigated. There were no serious adverse events related to AdPEDF.11 and no dose-limiting toxicities. Signs of mild, transient intraocular inflammation occurred in 25% of patients, but there was no severe inflammation. Six patients experienced increased intraocular pressure that was easily controlled by topical medication. All adenoviral cultures were negative. At 3 and 6 months after injection, 55 and 50%, respectively, of patients treated with 10(6)-10(7.5) PU and 94 and 71% of patients treated with 10(8)-10(9.5) PU had no change or improvement in lesion size from baseline. The median increase in lesion size at 6 and 12 months was 0.5 and 1.0 disk areas in the low-dose group compared with 0 and 0 disk areas in the high-dose group. These data suggest the possibility of antiangiogenic activity that may last for several months after a single intravitreous injection of doses greater than 10(8) PU of AdPEDF.11. This study provides evidence that adenoviral vector-mediated ocular gene transfer is a viable approach for the treatment of ocular disorders and that further studies investigating the efficacy of AdPEDF.11 in patients with neovascular AMD should be performed.