Heterologous expression and function evaluation of Gloeobacter violaceus rhodopsin in Escherichia coli / 生物工程学报

Proton-pumping rhodopsin (PPR) is a simple photosystem widely distributed in nature. By binding to retinal, PPR can transfer protons from the cytoplasmic to the extracellular side of the membrane under illumination, creating a proton motive force (PMF) to synthesize ATP. The conversion of light into chemical energy by introducing rhodopsin into nonphotosynthetic engineered strains could contribute to promoting growth, increasing production and improving cell tolerance of microbial hosts. Gloeorhodopsin (GR) is a PPR from Gloeobacter violaceus PCC 7421. We expressed GR heterologously in Escherichia coli and verified its functional activity. GR could properly function as a light-driven proton pump and its absorption maximum was at 539 nm. We observed that GR was mainly located on the cell membrane and no inclusion body could be found. After increasing expression level by ribosome binding site optimization, intracellular ATP increased, suggesting that GR could supply additional energy to heterologous hosts under given conditions.

Mutation analysis of pathogenic genes in a Henan family affected with congenital stationary night blindness / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect genetic mutations associated with autosomal dominant congenital stationary night blindness (ADCSNB) in a family from Henan province.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples of 14 family members. Based on 3 genes reported previously, PCR primers were designed and corresponding exons containing the mutation sites were amplified with PCR. PCR products were purified and directly sequenced.</p><p><b>RESULTS</b>A c.281C>T heterozygous missense mutation was detected in RHO gene in all of the patients. This mutation can cause a change of the protein structure (p.Thr94Ile). The same mutation was not detected in normal individuals from the family and 50 normal controls.</p><p><b>CONCLUSION</b>A c.281C>T mutation in RHO gene is responsible for the onset of ADCSNB in this Chinese family and results in symptoms of night blindness.</p>

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Family A G-Protein coupled receptors: an overview of structure-function relationships

Family A G-protein coupled receptors (AGPCRs) form the largest group of correlate receptors whose structure, a bundle of seven-trans-membrane (7 TM) helices, may be activated thus becoming able to transduce a signal from the extracellular medium to the cytosol. This activation may be constitutional, for instance due to permanent structural modifications, or be physiologically triggered by agonist binding at an external and accessible specific site. Based on thestructures of agonists, AGPCRs may be divided according to pharmacological assays into many classes of receptors, each one comprising many types or sub-types of proteins, as differentiated by specific binding of inhibitors, all of them performing a multitude of functions. It is noteworthy that AGPCRs have been more recently cloned and their sequences of amino acids determined in a large scale, a condition that has allowed these receptors to be sorted by a new criterium. Sequence analyses have consistently matched functional assays for classification of AGPCRs except for a certain number of functionally unknown receptors which have been cataloged as orphan receptors. A colossal number of AGPCRs, more than 10,000 sequences belonging to more than 1,000 different types of receptors, may nowadays be multiply-aligned what has been enabling the determination of parameters of residue conservation and characterization of special motifs along the structure of these proteins. There are at the present time, high-resolution 3D structures for the following AGPCRs: inactive rhodopsin, retinal-free opsin, Beta adrenoceptor and adenosine receptors. Among them, hodopsin structures are reliable enough to be used as prototypes for analyses of residue conservation and mechanisms of activation of receptors, specially at the level of the more conserved structure in the cytosolic half of their 7TM bundle.

Aproximaciones de bioquímica clásica al estudio de la relación entre la estructura y la función de la rodopsina Y / Approaches to the study of classical biochemistry of the relationship between structure and function of the rhodopsin

La proteína fotorreceptora rodopsina (R) fue extraída de los segmentos externos de los bastoncillos de retinas bovinas con el detergente n-dodecil β-D-maltósido (DM) y purificada a homogeneidad mediante cromatografía de afinidad. El entrecruzamiento químico de la R y de la rodopsina fotoactivada (R*) con los agentes bifuncionales sulfo-succinimidilo 4-(N-maleimidometilo) ciclohexano-1-carboxilato (sulfo-SMCC) o m-maleimidobenzoilo-N-hidroxisuccinimido ester, sugirieron la naturaleza oligomérica de la proteína fotorreceptora. La caracterización de los parámetros hidrodinámicos de la R y la R* en presencia de 0.1% DM, mediante cromatografía de exclusión molecular y sedimentación sobre gradientes de sacarosa, permitió estimar los tamaños de los complejos R:DM y R*: DM. Los resultados concuerdan con una estructura cuaternaria dimérica tanto para la R como para la R*. La R entrecruzada con sulfo-SMCC, en presencia de luz, fue estabilizada en un fotointermediario que absorbió a ~ 470 nm. Experimentos de proteólisis con termolísina sobre los dímeros nativos de R y sobre los monómeros de R generados por medio del uso de altas concentraciones de DM, complementados con estudios de modelaje basados en la estructura cristalina reportada de la proteína, sugirieron que el reactivo sulfo-SMCC generó un entrecruzamiento intramolecular entre la Cys140 y la Lys248 de la R, el cual posiblemente es el responsable de la incapacidad de la proteína de sufrir el cambio conformacional requerido para llegar a su estado fotoactivado.

Association between a new 3q; 5q chromosomal translocation and dystrophy of human retinal pigment epithelium

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degeneration. This group of disorders essentially leads to blindness due to mutations in different genes. The genetic basis affected by sporadic and inherited autosomal dominant, autosomal recessive or X-linked mutations is complex. In humans, RP is in most cases associated with missense mutations in the rhodopsin gene (RHO). RHO plays an important role in phototransduction pathways. So far, few studies have described associations between chromosomal alterations and RP. In this study, we present a case report of a premature, 32-week-old male baby who suffered from retinopathy, facial dysmorphisms and other disorders. His chromosomes were analyzed by conventional and high-resolution chromosomal techniques. This analysis revealed structural aberrations on chromosomes 3 and 5 with an apparently balanced chromosomal translocation with karyotype 46,XY,t(3;5)(q25;q11.2). Remarkably, the 3q breakpoint on the long arm of chromosome 3 is located close to the physical RHO chromosomal gene location. In this study, we describe presumably for the first time a possible association between a 3q;5q chromosomal alteration and RP. We conclude that the new detected chromosomal translocation may lead either to loss or inactivation of the intragenic RHO gene or its respective gene regulatory region. As a consequence, the chromosomal aberration may be responsible for retinitis pigmentosa.

Effects of Zinc and Hypothermic Process during the Light and Dark Adaptation of Vertebrate Retina

PURPOSE: The purpose of this study was to clarify the effects of zinc treatment and hypothermia on visual adaptation and visual sensitivity in bullfrogs (Rana catesbeiana), which are poikilothermal animals capable of adjusting quickly to environmental temperature changes. METHODS: The effects of both zinc treatment and hypothermia on visual sensitivity were studied by using electroretinogram (ERG) recording and absorption spectra scanning before and after zinc and TSQ (N-[6-methoxy-8-quinolyl]-p-toluene sulfonamide) treatment, with or without temperature changes. RESULTS: In spite of malnutrition due to hibernation, the optimal zinc concentration effect was obtained at 10-4 M (10-2 M 200 microliter ZnCl2 in 20 microliter Ringer's solution) according to ERG recording. After zinc treatment and hypothermia induction, increments of all ERG components and thresholds were taken by ERG recording. These results showed that both zinc treatment and hypothermia may increase visual sensitivity during visual adaptation. In spectral scans, the absorbance increment due to zinc treatment and hypothermia was shown over the whole spectral range (400~750 nm), and it was especially prominent at alpha-peak (about 500 nm). In addition, there was a decrease in absorption differences between dark adaptation and light adaptation after zinc treatment. Furthermore, according to the visual sensitivity decrement using TSQ as a zinc specific chelator, this visual sensitivity increase was shown to be caused by zinc. CONCLUSIONS: As the results suggest, both zinc treatment and hypothermic effects may improve visual sensitivity by promoting rhodopsin regeneration and inhibiting rhodopsin bleaching induced by light illumination. Zinc may activate the enzyme activity of retinol dehydrogenase and phosphodiesterase, while hypothermic effects may improve precursor transport, which is required for rhodopsin regeneration, by tightening membrane adhesion between retinas and retinal pigment epithelia. In addition, we believe that zinc treatment and hypothermic effects may work synergistically to accelerate visual sensitivity during visual adaptation.

Clinical features of autosomal dominant retinitis pigmentosa associated with a Rhodopsin mutation

<p><b>INTRODUCTION</b>Retinitis pigmentosa (RP) describes a group of inherited disorders characterised by progressive retinal dysfunction, cell loss and atrophy of retinal tissue. RP demonstrates considerable clinical and genetic heterogeneity, with wide variations in disease severity, progression, and gene involvement. We studied a large family with RP to determine the pattern of inheritance and identify the disease-causing mutation, and then to describe the phenotypic presentation of this family.</p><p><b>MATERIALS AND METHODS</b>Ophthalmic examination was performed on 46 family members to identify affected individuals and to characterise the disease phenotype. Family pedigree was obtained. Some family members also had fundus photographs, fluorescein angiography, and/or optical coherence tomography (OCT) analysis performed. Genetic linkage was performed using short tandem repeat (STR) polymorphic markers encompassing the known loci for autosomal dominant RP. Finally, DNA sequencing was performed to identify the mutation present in this family.</p><p><b>RESULTS</b>Clinical features included nyctalopia, constriction of visual fields and eventual loss of central vision. Sequence analysis revealed a G-to-T nucleotide change in the Rhodopsin gene, predicting a Gly-51-Val substitution.</p><p><b>CONCLUSIONS</b>This large multi-generation family demonstrates the phenotypic variability of a previously identified autosomal dominant mutation of the Rhodopsin gene.</p>

A recurrent rhodopsin gene missense mutation in a Chinese family with autosomal dominant retinitis pigmentosa / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect mutation in the rhodopsin gene (RHO) in a Chinese family with autosomal dominant retinitis pigmentosa (ADRP).</p><p><b>METHODS</b>A total of 25 family members from a Chinese family were investigated. All the subjects were examined clinically by direct funduscopy, perimetry and vision test. Evaluation of the proband included electroretinography (ERG). Genomic DNA was extracted using standard method. The complete coding regions of RHO were amplified by polymerase chain reaction (PCR) and the PCR products were subjected to automatic DNA sequencing.</p><p><b>RESULTS</b>512 C>T (P171L), a recurrent missense mutation was detected in the proband. All 12 affected subjects in the family were heterozygous for the mutation. The affected individuals had night blindness at the age of 5-6 years. They had relatively severe impairment of visual acuity and suffered a gradual loss of peripheral visual field at the age of 20-30 years. And they went blind at the age of 40-50 years. Rod and cone ERG were not detectable in the proband.</p><p><b>CONCLUSION</b>A recurrent missense mutation, 512C>T (P171L), was detected in a Chinese family with ADRP.</p>

A complete screen for mutations of the rhodopsin gene in a panel of Chinese patients with autosomal dominant retinitis pigmentosa / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the prevalence of rhodopsin (RHO) mutations and the genotype-phenotype relationships in Chinese patients with autosomal dominant retinitis pigmentosa (ADRP) by conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing.</p><p><b>METHODS</b>We have screened the five coding exons and splice sites of RHO gene in 27 probands who had no relativity from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using CSGE and direct DNA sequencing. Family members of some probands with disease-associated mutations were also genotyped to determine whether the RHO mutations segregated with retinitis pigmentosa (RP) in their families.</p><p><b>RESULTS</b>Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified separately in two families, thus the frequency of RHO mutations among this set of Chinese ADRP families is about 7.4% (2/27). Pro347Leu mutation was found in one ADRP proband as well as three her children who also had RP. She had relatively early onset at about 17 years. The only one child without this mutation had no symptom or sign of RP at age of 34. Pro327 (1-bp del) was identified in a late-onset ADRP patient, who appeared night blindness around 30 years old and in her fifties electroretinogram (ERG) has been flat in both scotopic and photopic phases. Family analysis showed that this mutation also existed in her younger daughter and her elder sister, both of them also had RP. Three other family members were genotypically and phenotypically normal. Neither of the two mutations was detected in 100 normal controls.</p><p><b>CONCLUSIONS</b>The frequency of RHO mutations in Chinese patients was lower than that in Europe and North America. The phenotype of the patients with Pro347Leu corresponded to type 1 ADRP, with severe rod degeneration and some cone preservation later, while the phenotype of the patients carrying Pro327 (1-bp del) corresponded to type 2 ADRP, with a concomitant loss of rod and cone visual function. CSGE was found to be a sensitive, simple, and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.</p>

Identification of functionally important acidic residues in transducin by group-specific labeling

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50 per cent inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.

Screening of candidate genes in a family with autosomal dominant retinitis pigmentosa / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the causative mutation in a 5 generation pedigree with autosomal dominant retinitis pigmentosa (ADRP).</p><p><b>METHODS</b>Genomic DNA from four patients and 4 normal persons in the same pedigree suffering ADRP were extracted, and subsequently eight exons of three ADRP candidate genes were screened for mutations by a combined polymerase chain reaction-single strand conformation polymorphism and DNA sequencing techniques.</p><p><b>RESULTS</b>A new point mutation in rhodopsin gene at codon 52 of exon 1 (TTC to TAC) that resulted in a substitution of Tyr to Phe was detected in the four affected family members, but not in the four control individuals from the same pedigree.</p><p><b>CONCLUSION</b>A causative mutation of rhodopsin gene was identified in a large Chinese pedigree with ADRP. The present study confirmed the molecular genetic heterogeneity of ADRP.</p>

Biological role of melatonin on retinal rhodopsin. an experimental study

Melatonin plays a significant role in enhancing the immune system, preventing cancer by protecting DNA, protecting human white blood cells from radiation and has a direct protective effect on the heart and the circulatory system in general. It also has anticataract function and reduces lipid peroxidation. In the present work, the effect of treatment with different doses of exogenous melatonin on the biophysical characteristics of retinal rhodopsin at different periods was studied using different techniques. New Zealand white rabbits were grouped into five sets [I, II, III, IV and V]. Animals of set I was used as control. Sets II, III, IV and V were injected daily with melatonin by doses of 10, 100, 500 and 1000 mu g, respectively, for the periods of 24 hr, one, three and five months. After the studied periods, the retinas were separated and rhodopsin was extracted, then the following measurements were carried out: Measurement of the uv-visible absorption spectrum, estimation of total protein content and determination of the amino acid pattern. The data indicated that melatonin at low doses [10,100 mug] significantly affects both total protein and the opsin part rather than the high doses [500, 1000 mu g]. Low doses of melatonin will preserve all the amino acids, which are the most important for rhodopsin structure and characteristics. Melatonin at physiological doses - similar to those secreted in the body- is useful for the eye but at high doses melatonin may change the structure of rhodopsin molecule

Retinal rhodopsin of rabbits after treatment with different doses of melatonin

Recently, melatonin is a broad-spectrum antioxidant for both animals and plants. It prevents oxidative damage at the level of cells, tissues, organs and organisms. In the present study, the effect of different doses of melatonin [10, 100, 500 and 1000 mu g] on the retinal rhodopsin was studied after different periods of 24 hr, one, three and five months. The rabbits were injected with melatonin daily early in the morning. After the studied periods, rhodopsin was extracted then the following measurements were carried out on it; purification of rhodopsin using a column of calcium triphosphate and celite; electrophoretic separation using SDS-polyacrylamide gel and dielectric measurements in the frequency range of 105 - 107 Hz. The obtained results indicated that there was no change in the molecular weight of rhodopsin at different doses and periods. The dipole moment was decreased at low doses and increased at high doses of melatonin. From the obtained results, it was concluded that melatonin at physiological doses could be useful for the eye

Screening for point mutations in rhodopsin gene among one hundred Chinese patients with retinitis pigmentosa / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To test the frequency and pattern of rhodopsin (RHO) mutations in Chinese retinitis pigmentosa (RP) patients and to evaluate their effects in the pathogenesis of RP.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples of 100 Hong Kong Chinese RP patients. Sequence variants of the entire coding exons of the RHO gene were tested using PCR, conformation sensitive gel electrophoresis and DNA sequencing.</p><p><b>RESULTS</b>Totally six nucleotide changes were identified, among which three were silent mutations, two missense mutations and one deletion mutation. P347L was found in one RP proband and her three children who also had RP. P327(1 bp del) was novel and detected in a late-onset RP patient of 53 years. Her 26-year-old daughter, also carrying the identified mutation, had no RP phenotypes except for the mottled retinal pigment epithelium (RPE) revealed by fundal examination. Neither of the two mutations was detected in normal controls.</p><p><b>CONCLUSION</b>Two patients had disease-causing mutations in the RHO gene, thus RHO mutations cause about 2.0% (95% confidence interval: 0.2%-7.0%) of all RP among Chinese in Hong Kong. A highly conserved C-terminal sequence QVS(A)PA was altered due to P347L and thereby resulting in an aberrant subcellular localization of rhodopsin. Loss of all six phosphorylatable residues at the C-terminus and the highly conserved C-terminal sequence QVS(A)PA may occur because of P327(1 bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying P327(1 bp del) would be of greatest value.</p>

Mutation analysis of codons 345 and 347 of rhodopsin gene in Indian retinitis pigmentosa patients.

More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P --> S/A/R/Q/L/T) were absent while the codon 345 mutation V --> M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.

Differentiation of Recoverin Immunoreactive Cone Bipolar Cells and Their Timing of Synaptic Formation with GABAergic Amacrine Cells in the Rat Retina / 대한해부학회지

Recoverin is a member of the large family of EF-hand calcium binding proteins (Baimbridge et al., 1992), and it is thought to be involved in the regulation of phosphodiesterase in photoreceptors and in the phosphorylation of activated rhodopsin (Polans et al., 1996). Although the functional significance of recoverin in cone bipolar cells is not fully understood, the antiserum against recoverin has been widely used to identify a certain population of cone bipolar cells (Milam et al., 1993; Sasso's Pognetto et al., 1994; Euler & W sle, 1995). GABA is well known to act as major neurotransmitters in the mammalian central nervous system including retina. This study was conducted to identify the development process of recoverin-labeled cone bipolar cells, and the timing points of synaptic formation of the labeled bipolar cells and GABAergic amacrine cells in the rat retina. The results were as follows; In the adult rat retina, recoverin-labeled cone bipolar cells were subdivided into twotypes; type 2 cells with axon terminal stratified in sublamina a of the inner plexiform layer (IPL), and type 8 cells with axon terminals stratified in sublamina b of the IPL. Recoverin-labeled cone bipolar cells began to appear from postnatal day 5. The axon terminals of recoverin-labeled type 2 cone bipolar cells stratified at postnatal day 10, while those of type 8 cone bipolar cells stratified at postnatal day 13. The axon terminals of type 2 cone bipolar cells made ribbon synapses onto GABAergic amacrine cells in the IPL at postnatal day 10. These results demonstrate that recoverin-labeled type 2 cone bipolar cells differentiate earlier than recoverin-labeled type 8 cone bipolar cells, and suggest that GABAergic amacrine cells may play important roles in visual processing of recoverin-labeled type 2 cone bipolar cells by making synapse onto these cells at early stage. Synapses between type 2 cone bipolar cells and GABAergic amacrine cells are formed about the time of postnatal day 10 for visual processing.

Effect of detergents and lipids on transducin photoactivation by rhodopsin

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1 per cent digitonin, following rod outer segment (ROS) solubilization with 1 per cent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), WAS used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1 per cent digitonin following ROS solubilization with 1 per cent digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2 per cent n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1 per cent CHAPS or 1 per cent DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075 per cent egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximun amount of GTP by transducin at 0.0075 per cent PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15 per cent PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

The parathyroid hormone-2 receptor: Current status

G-protein coupled receptors form a large superfamily of plasma membrane proteins which serve a variety of signal transduction roles. New receptors continue to be identified. Based on sequence homology the superfamily can currently be divided into three families, the rhodopsin family which includes the vast majority of identified receptors, and the secretin. and metabotropic glutamate receptor families which share a general architecture with each other and the rhodopsin family but no obvious sequence identity. Screening for additional members of the secretin family led to the identification of the parathyroid hormone-2 (PTH2) receptor. Ligand recognition by the PTH2 receptor partially overlaps that of the PTH/parathyroid hormone-related peptide (PTHrP) receptor. This has facilitated structure-function analysis of ligands for these receptors. The physiological role of the PTH2 receptor is under investigation but its distribution suggests that it may be a neurotransmitter receptor and could participate in modulation of a number of organ systems. The relative abundance of PTH2 receptor mRNA in the brain and the inability to detect mRNA encoding PTH, its only currently identified ligand, suggest the existence of another endogenous ligand, for which evidence has recently been obtained.