Biomedical subjects

K Palczewski

Publications and source records attributed to K Palczewski.

At least 55 records · Page 3Linked to original sources

PubMed1999

A novel form of rhodopsin kinase from chicken retina and pineal gland.

The G protein-coupled receptor kinases (GRKs) are important enzymes in the desensitization of activated G protein-coupled receptors (GPCR). Seven members of the GRK family have been identified to date. Among these enzymes, GRK1 is involved in phototransduction and is the most specialized kinase of the family. GRK1 phosphorylates photoactivated rhodopsin (Rho*), initiating steps in its deactivation. In this study, we found that chicken retina and pineal gland express a novel form of GRK that has sequence features characteristic of GRK1. However, unlike bovine GRK1 which is farnesylated, chicken GRK1 contains a consensus sequence for geranylgeranylation. Peptides corresponding to the C-terminal sequence of chicken GRK1 are geranylgeranylated by a cytosolic extract of chicken liver. Based on results of molecular cloning and immunolocalization, it appears that both rod and cone photoreceptors express this novel GRK1. These data indicate a larger sequence diversity of photoreceptor GRKs than anticipated previously.

Amino Acid Sequence↗

PubMed1999

Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.

Amino Acid Sequence↗

PubMed1999

beta-arrestin-dependent desensitization of luteinizing hormone/choriogonadotropin receptor is prevented by a synthetic peptide corresponding to the third intracellular loop of the receptor.

Desensitization is a ubiquitous response of guanine nucleotide-binding protein-coupled receptors (GPCRs) characterized by the waning of effector activity despite continued presence of agonist. Binding of an arrestin to the activated, often phosphorylated GPCR triggers desensitization. We reported for the luteinizing hormone/choriogonadotropin receptor (LH/CG R) that beta-arrestin tightly bound to porcine ovarian follicular membranes mediates agonist-dependent desensitization of LH/CG R-stimulated adenylyl cyclase (AC) activity (Mukherjee, S., Palczewski, K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and Hunzicker-Dunn, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 493-498). We now show that addition of a synthetic peptide corresponding to the entire third intracellular loop (3i) of the LH/CG R completely and specifically reverses desensitization of AC activity, with an ED50 of 10 microM but does not modulate basal, hCG-stimulated, or forskolin-stimulated AC activities. beta-Arrestin binds selectively to the 3i peptide coupled to activated Sepharose. Desensitization of LH/CG R-stimulated AC activity is rescued when the 3i peptide is preincubated with exogenous beta-arrestin. These results show that endogenous beta-arrestin participates in cell-free desensitization of agonist-dependent LH/CG R-stimulated AC activity in follicular membranes by interacting directly with the 3i loop of the receptor, thereby preventing Gs activation.

Amino Acid Sequence↗

PubMed1999

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein.

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.

Acyltransferases↗

PubMed1999

Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily.

The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca2+]free and inhibit GCs when [Ca2+]free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca2+-binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC.

Amino Acid Sequence↗

PubMed1999

Light inhibition of bovine retinal rod guanylyl cyclase mediated by beta gamma-transducin.

Photoreceptor guanylyl cyclase (ROS-GC), converting GTP into cGMP and pyrophosphate, is a key enzyme in the regulation of the visual transduction cascade. ROS-GC requires GC-activating proteins (GCAPs) and low free [Ca] for full activity. We found that when choline or potassium were the major cations present, light caused a 70% inhibition of stimulated ROS-GC in native unstripped membranes. In the presence of sodium ions, however, no inhibition was observed. ROS-GC activity of ROS membranes, stripped of transducin and other components, was not affected by light when reconstituted with GCAP1 only. However, when stripped ROS membranes were reconstituted with both GCAP1 and either transducin (T alpha beta gamma) or the T beta gamma-subunits, the inhibition of ROS-GC by light was restored. The T alpha-subunit alone was ineffective. These results suggest that under saturating light conditions, ROS-GC may be regulated by T beta gamma and cations, providing a possible mechanism of desensitization and light adaptation.

Animals↗

PubMed1999

Identification of a guanylyl cyclase-activating protein-binding site within the catalytic domain of retinal guanylyl cyclase 1.

Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide,968GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between alpha-helix 3 and beta-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with Gs alpha and Gi alpha, respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.

Amino Acid Sequence↗

PubMed1999

A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes.

The luteinizing hormone/choriogonadotropin (LH/CG) receptor (R) is a heptahelical R that, upon agonist binding, activates the stimulatory guanine nucleotide-binding protein (Gs) and the downstream effector adenylyl cyclase (AC). Like other G protein-coupled Rs, the LH/CG R subsequently exhibits reduced agonist-dependent effector activity, or desensitization, in response to saturating agonist. Unlike desensitization of many other G protein-coupled Rs, the in vivo desensitization response of LH/CG R-stimulated AC activity of ovarian follicles to the preovulatory surge of LH can be mimicked under cell-free conditions. Based on evidence that porcine ovarian follicular membranes unexpectedly contained beta-arrestin-1, the role of arrestins in desensitization of the LH/CG R was investigated. Results showed that neutralizing arrestin antibodies blocked the development of desensitization and that desensitization was rescued with a synthetic peptide corresponding to the antibody-binding epitope on beta-arrestin-1. These results suggest that endogenous beta-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R. Addition of recombinant purified beta-arrestin-1 mimicked human chorionic gonadotrophin to promote desensitization of human chorionic gonadotrophin-stimulated AC activity, in the presence of the ATP phosphorylation antagonist adenylyl-imidodiphosphate, with an ED50 of approximately 0.1 nM. Increased levels of an 87-kDa protein reactive with glycoprotein hormone R-reactive antibody, consistent with the LH/CG R, coimmunoprecipitated with follicular membrane beta-arrestin-1 in response to LH/CG R activation compared with unactivated R. Taken together, these results show that ovarian follicles contain membrane-associated beta-arrestin-1, that beta-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R, and that the trigger for beta-arrestin-1 binding to the LH/CG R appears to be R activation.

Adenylyl Imidodiphosphate↗

PubMed1999

Molecular mechanism of visual transduction.

Our vision renders an incredible wealth of information about the external environment presented in the form of light of different wavelengths and intensities. To operate in a wide range of light intensities, our visual system has developed several mechanisms that allow an adjustment of its sensitivity to light. Immense progress has been made in understanding how light is captured and activates visual phototransduction cascade within photoreceptor cells; however, much less is known about desensitization. It has been known for some time, that many of these processes rely on Ca2+ as the principal modifier of phototransduction. Ca(2+)-binding proteins (CBPs) are specifically poised to take advantage of transient changes in [Ca2+] to act as enzymatic regulators. Some other CBPs are capable of changing the intracellular Ca2+ buffering capacity. Various retinal CBP proteins have been identified, including recoverin, GCAP1, GCAP2, GCAP3, GCIP, CBP1, CBP3 and CBP4. Although these numerous CBPs were identified, functions can be ascribed to only a few of them. Recently, genetic, physiological and biochemical analyses of retinal diseases have yielded additional insights into the role of many phototransduction proteins, including CBPs. Understanding the properties and the functions of these CBPs will pave the way for a more complete picture of visual transduction and accompanying desensitization processes.

Animals↗

PubMed1998

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin.

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.

Animals↗

PubMed1998

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal.

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.

Alcohol Oxidoreductases↗

PubMed1998

Calcium-sensitive particulate guanylyl cyclase as a modulator of cAMP in olfactory receptor neurons.

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events.

Animals↗

PubMed1998

High expression levels in cones of RGS9, the predominant GTPase accelerating protein of rods.

RGS9 is a member of the RGS family of GTPase accelerating proteins (GAPs) for heterotrimeric G proteins. We have explored its contribution to GTPase acceleration in mammalian rod and cone photoreceptors. When RGS9 was specifically removed from detergent extracts of rod outer segments by immunodepletion, the extracts lost nearly all of their GAP activity stimulatable by the inhibitory subunit of cGMP phosphodiesterase. Immunolocalization using monoclonal antibodies and confocal microscopy revealed that RGS9 is present in cones at significantly higher levels than in rods. Thus, RGS9 is the predominant source of GAP activity in rod outer segments, and RGS9 concentration emerges as a potentially important determinant of the faster response kinetics and lower sensitivity of mammalian cones, as compared with rods.

Animals↗

PubMed1998

Guanylate-cyclase-inhibitory protein is a frog retinal Ca2+-binding protein related to mammalian guanylate-cyclase-activating proteins.

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+-binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+-binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.

Amino Acid Sequence↗

PubMed1998

Molecular forms of human rhodopsin kinase (GRK1).

The G protein-coupled receptor kinases (GRKs) are critical enzymes in the desensitization of activated G protein-coupled receptors. Six members of the GRK family have been identified to date. Among these enzymes, GRK1 (rhodopsin kinase) is involved in phototransduction and is the most specialized of the family. GRK1 phosphorylates photoactivated rhodopsin, initiating steps in its deactivation. In this study, we found that human retina expressed all GRKs except GRK4. Based on results of molecular cloning and immunolocalization, it appears that both rod and cone photoreceptors express GRK1. This conclusion was supported by the cloning of only GRK1 from cone-dominated chicken retina. Human photoreceptors also transcribe a splice variant of GRK1, which differs in its C-terminal region next to the catalytic domain. This novel variant, GRK1b, is produced by retention of the last intron. mRNA encoding GRK1b is exported to the cytosol; however, the level of the protein is relatively low compared with GRK1 (now called GRK1a), and GRK1b appears to have very low catalytic activity. Thus, these studies suggest that rods and cones, express the same form of GRK1.

Alternative Splicing↗

PubMed1998

A null mutation in the photoreceptor guanylate cyclase gene causes the retinal degeneration chicken phenotype.

The retinas of the retinal degeneration (rd) chicken are fully developed and possess normal morphology at hatching but fail to respond to light stimulation. Analyses of retinal cGMP, the internal messenger of phototransduction, show that the amount of cGMP in predegenerate, fully developed rd/rd photoreceptors is 5-10 times less than that seen in normal photoreceptor cells. We show that the low levels of cGMP in rd chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. Thus, the rd chicken is a model for human Leber's congenital amaurosis. Absence of GC1 in rd retina prevents phototransduction and affects survival of rods and cones but does not interfere with normal photoreceptor development.

Amino Acid Sequence↗

PubMed1998

Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

Amino Acid Sequence↗

PubMed1998

Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man.

Rhodopsin kinase (RK), a specialized G-protein-coupled receptor kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.

Animals↗