916 Differential regulation of choline acetyltransferase expression in the central nervous system of Drosophila: A study using temperature-sensitive mutants (original) (raw)
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Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments
The Journal of General Physiology, 1989
Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition.
Photoreceptor guanylate cyclases: a review
Bioscience reports, 1997
Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca2+ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca2+ signals. The low Ca2+ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca2+ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section...
Photoreceptor specific guanylate cyclases in vertebrate phototransduction
Guanylate Cyclase, 2002
Two membrane bound guanylate cyclases are expressed in vertebrate photoreceptor cells. They serve a key function in photoreceptor physiology as they synthesize the intracellular transmitter of photoexcitation guanosine 3′,5′-cyclic monophosphate (cGMP). Both cyclases named ROS-GC1 and ROS-GC2 form a subclass of membrane bound cyclases and differ in many aspects from hormone peptide receptor guanylate cyclases. One unique feature is their regulation by three small Ca 2+-binding proteins called GCAPs. These regulatory proteins sense changes in the cytoplasmic Ca 2+-concentration [Ca 2+ ] during illumination and activate ROS-GCs when the [Ca 2+ ] decreases below the value in a dark adapted cell of 500-600 nM. Recent work has identified the target regions of GCAP-1 in ROS-GC1. In addition to GCAPs several other proteins including aktin, tubulin, a glutamic-acid-rich protein and a GTPase accelerating protein (RGS9) were found to interact with ROS-GC1 and probably form a multiprotein complex. (Mol Cell Biochem 230: 97-106, 2002)
Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones
Frontiers in Molecular Neuroscience, 2014
In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca 2+ sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of GC activating proteins (GCAPs) and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca 2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca 2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca 2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca 2+ affinity, is recruited later during the course of the photon response as Ca 2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.
The EMBO Journal, 1991
The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key events leading to the reopening of cGMP-gated channels after photoexcitation. Guanylate cyclase activity in vertebrate rod outer segments is dependent on the free calcium concentration. The basal activity of the enzyme observed at high concentrations of free calcium (>0.5 /M) increases when the free calcium concentration is lowered into the nanomolar range (< 0.1 /tM). This effect of calcium is known to be mediated by a soluble calcium-sensitive protein in a highly cooperative way. We here show that this soluble protein, i.e. the modulator of photoreceptor guanylate cyclase, is a 26 kd protein. Reconstitution of the purified 26 kd protein with washed rod outer segment membranes containing guanylate cyclase revealed a 3to 4-fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 ,tM to 4 nM. Guanylate cyclase in whole rod outer segments was stimulated 10-fold in the same calcium range. The activation process in the reconstituted system was similar to the one in the native rod outer segment preparation, it showed a high cooperativity with a Hill coefficient n between 1.4 and 3.5. The half-maximal activation occurred between 110 and 220 nM free calcium. The molar ratio of the modulator to rhodopsin is 1:76 32. The protein is a calcium binding protein as detected with 45Ca autoradiography. Partial amino acid sequence analysis revealed a 60% homology to visinin from chicken cones.
Visual Consequences of Molecular Changes in the Guanylate Cyclase-Activating Protein
PURPOSE. We characterized and modeled changes in visual performance associated with a Tyr99Cys mutation in guanylate cyclase-activating protein-1 (GCAP1) in four family members aged between 39 and 55 years old. Guanylate cyclase and its activating protein are molecules in the visual transduction pathway that restore cyclic GMP (cGMP) following its lightactivated hydrolysis. The mutation causes an excess of cGMP in the dark and results in progressive photoreceptor loss.
Journal of Biological Chemistry, 1999
The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca 2؉ ]. GCAP-1 is a Ca 2؉binding protein and belongs to the superfamily of EFhand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15amino acid peptide located adjacent to the second EFhand motif (Phe 73-Lys 87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe 73-Lys 87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca 2؉ ]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.
The Photoreceptor Guanylate Cyclase Is an Autophosphorylating Protein Kinase
Journal of Biological Chemistry, 1996
The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATP␥S (adenosine 5-O-(3-thiotriphosphate)), AMPPNP (5-adenylyl ,␥-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N 3 [␣-32 P]ATP. The enzyme has a protein kinase activity that is Mg 2؉-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The K m for ATP is 81 M. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13acetate/L-␣-phosphatidylserine/Ca 2؉ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.
European Journal of Biochemistry, 1998
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 Ca 2ϩ -binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca 2ϩ ], but inhibits GC in high [Ca 2ϩ ], 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 Ca 2ϩ -binding motif of GCIP is disabled for Ca 2ϩ 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 Ca 2ϩ -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 [Ca 2ϩ ], competing with GCAP1 and GCAP2 for GC regulatory sites.
Localization of guanylate cyclase-activating protein 2 in mammalian retinas
Proceedings of the National Academy of Sciences, 1997
Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca 2+ , a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC ...