Purification of rhodopsin kinase by recoverin affinity chromatography.
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Animals↗
Biomedical subjects
J B Hurley
Publications and source records attributed to J B Hurley.
Heterologous expression and assays for photoreceptor guanylyl cyclases and guanylyl cyclase activating proteins.
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Animals↗
Biochemical analysis of phototransduction and visual cycle in zebrafish larvae.
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Animals↗
Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.
Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.
Amino Acid Sequence↗
RET-RGS, a retina-specific regulator of G-protein signaling, is located in synaptic regions of the rat retina.
RGS (regulators of G protein signaling) proteins negatively regulate the alpha subunit of G proteins by accelerating their intrinsic GTPase activity. In a previous work, we reported the cloning of a cDNA encoding for a new RGS protein, RET-RGS. We showed that it is specifically expressed in the retina, notably by photoreceptor cells and that it has an in vitro GAP activity on transducin. To understand the role of RET-RGS, and in particular to determine whether it regulates the phototransduction cascade in photoreceptor cells, RET-RGS was immunolocalized on rat retina sections. Whereas no labeling was detected in outer nor inner segments of photoreceptors cells, dense immunoreactive products were localized in the outer and inner plexiform layers which correspond to the regions of synaptic interplay between the different neurons of the retina including the photoreceptor cells. These results rule out a role of RET-RGS on the phototransduction cascade and suggest that it may participate in retina specific synaptic transductions.
Animals↗
Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.
Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.
Amino Acid Sequence↗
Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases.
Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.
Amino Acid Sequence↗
Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.
Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/- rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/- mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/- mice raised in constant darkness. One day of constant light caused the rods in the RK-/- mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.
Animals↗
Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins.
Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.
Amino Acid Motifs↗
Carcinoids of the common bile duct: a case report and literature review.
Carcinoids of the extrahepatic bile ducts and particularly the common bile duct are extremely rare. A 65-year-old woman presented with obstructive jaundice. Laboratory and imaging studies gave results that were consistent with an obstructing lesion in the common bile duct. In this case, a stent was inserted initially to decompress the bile ducts. Subsequently a laparotomy and pancreaticoduodenectomy were performed and a tissue diagnosis of carcinoid of the common bile duct was made. The patient was well with no evidence of recurrence 17 months postoperatively. The authors believe this is the 19th reported case of an extrahepatic bile duct carcinoid.
Aged↗
The kinase homology domain of retinal guanylyl cyclases 1 and 2 specifies the affinity and cooperativity of interaction with guanylyl cyclase activating protein-2.
RetGC-1 and RetGC-2 are photoreceptor membrane guanylyl cyclases that are regulated by the Ca2+-binding protein, GCAP-2. We used a protease protection assay to localize regions of the intracellular domains of RetGCs important for the interaction with GCAP-2 and found that GCAP-2 reduces the access of trypsin to a site in the kinase homology domain (KHD) of RetGC-1. The protective effect of GCAP-2 is independent of Ca2+. We also found that RetGC-2 and GCAP-2 interact cooperatively with high affinity, but RetGC-1 and GCAP-2 interact noncooperatively with low affinity. By analyzing RetGC-1/RetGC-2 chimeras we demonstrated that the affinity and cooperativity of the interaction with GCAP-2 is dictated by the structure of the KHD. These findings suggest that GCAP-2 interacts constituitively with the KHDs of RetGC-1 and RetGC-2 and that cGMP synthesis is controlled by Ca2+-dependent conformational changes in the RetGC/GCAP complex.
Animals↗
Autophosphorylation and ADP regulate the Ca2+-dependent interaction of recoverin with rhodopsin kinase.
Recoverin is a 23 kDa myristoylated Ca2+-binding protein that inhibits rhodopsin kinase. We have used surface plasmon resonance to investigate the influences of Ca2+, myristoylation, and adenine nucleotides on the recoverin-rhodopsin kinase interaction. Our analyses confirmed that Ca2+ is required for recoverin to bind RK. Myristoylation had little effect on the affinity of recoverin for the kinase, but it raised the K0.5 for Ca2+ from 150 nM for nonacylated recoverin to 400 nM for myristoylated recoverin. Finally, our studies also revealed two separate and previously unreported effects of adenine nucleotides on the recoverin-rhodopsin kinase binding. The interaction is weakened by autophosphorylation of the kinase, and it is strengthened by the presence of ADP.
Adenosine Diphosphate↗
Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase.
Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.
Adenylyl Cyclases↗
Functional heterogeneity of transducin alpha subunits.
The N-terminal glycine of transducin alpha subunits is acylated by lauroyl (C12:0), myristoyl (C14:0), (cis-delta5)-tetradecaenoyl (C14:1) or (cis,cis-delta5,delta8)-tetradecadienoyl (C14:2) fatty acyl groups. We examined functional heterogeneity of transducin by sequentially eluting it from bleached outer segments using increasing concentrations of GTP then identifying the N-terminal acyl groups on the eluted alpha subunits. C14:2 acylated transducin eluted at low GTP concentrations followed by C12:0, C14:1 and C14:0 transducin at higher GTP concentrations. This suggests functional heterogeneity in the different forms of transducin alpha subunits.
Acetylation↗
Ca2+-dependent conformational changes in bovine GCAP-2.
GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.
Amino Acid Sequence↗
Zebrafish retinal mutants.
We have initiated a genetic analysis of the zebrafish visual system to identify novel molecules involved in vertebrate retinal function. Zebrafish are highly visual; they have four types of cones as well as rod photoreceptors, making it possible to study both rod and cone-mediated visual responses. To identify visual mutants, optokinetic responses of mutagenized larvae are measured in a three-generation screen for recessive mutations. By measuring visual behavior our genetic screen has been targeted towards identifying mutants that do not have gross morphological abnormalities. The electroretinogram (ERG) of optokinetic-defective mutants is recorded and their retinas are examined histologically to localize defects to the retina. In this report, we summarize our screening results and ERG and histological analyses of the five morphologically normal mutants we have analyzed to date. Additionally, the more detailed characterization of a red-blind mutant that we have isolated is summarized. Our results indicate that mutants with defects in various processes such as photoreceptor synaptic transmission, photoreceptor adaptation and cell-type specific survival and/or function can be identified using this approach.
Animals↗
Rhodopsin phosphorylation and its role in photoreceptor function.
Light-stimulated phosphorylation of rhodopsin was first described 25 years ago. This paper reviews the progress that has been made towards (i) understanding the nature of the enzymes that phosphorylate and dephosphorylate rhodopsin (ii) identifying the sites of phosphorylation on rhodopsin and (iii) understanding the physiological importance of rhodopsin phosphorylation. Many important questions related to rhodopsin phosphorylation remain unanswered and new strategies and methods are needed to address issues such as the roles of Ca2+ and recoverin. We present one such method that uses mass spectrometry to quantitate rhodopsin phosphorylation in intact mouse retinas.
Animals↗
Release of low molecular weight silicones and platinum from silicone breast implants.
We have conducted a series of studies addressing the chemical composition of silicone gels from breast implants as well as the diffusion of low molecular weight silicones (LM-silicones) and heavy metals from intact implants into various surrounding media, namely, lipid-rich medium (soy oil), aqueous tissue culture medium (modified Dulbecco's medium, DMEM), or an emulsion consisting of DMEM plus 10% soy oil. LM-silicones in both implants and surrounding media were detected and quantitated using gas chromatography (GC) coupled with atomic emission (GC-AED) as well as mass spectrometric (GC/MS) detectors, which can detect silicones in the nanogram range. Platinum, a catalyst used in the preparation of silicone gels, was detected and quantitated using inductive argon-coupled plasma/mass spectrometry (ICP-MS), which can detect platinum in the parts per trillion range. Our results indicate that GC-detectable low molecular weight silicones contribute approximately 1-2% to the total gel mass and consist predominantly of cyclic and linear poly-(dimethylsiloxanes) ranging from 3 to 20 siloxane [(CH3)2-Si-O] units (molecular weight 200-1500). Platinum can be detected in implant gels at levels of approximately 700 micrograms/kg by ICP-MS. The major component of implant gels appears to be high molecular weight silicone polymers (HM-silicones) too large to be detected by GC. However, these HM-silicones can be converted almost quantitatively (80% by mass) to LM-silicones by heating implant gels at 150-180 degrees C for several hours. We also studied the rates at which LM-silicones and platinum leak through the intact implant outer shell into the surrounding media under a variety of conditions. Leakage of silicones was greatest when the surrounding medium was lipid-rich, and up to 10 mg/day LM-silicones was observed to diffuse into a lipid-rich medium per 250 g of implant at 37 degrees C. This rate of leakage was maintained over a 7-day experimental period. Similarly, platinum was also observed to leak through intact implants into lipid-containing media at rates of approximately 20-25 micrograms/day/250 g of implant at 37 degrees C. The rates at which both LM-silicones and platinum have been observed to leak from intact implants could lead to significant accumulation within lipid-rich tissues and should be investigated more fully in vivo.
Breast Implants↗