Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism (original) (raw)
Related papers
Biochemistry, 1998
Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca 2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca 2+-binding proteins from the calmodulin superfamily, the Ca 2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca 2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca 2+-sensitive modulation of GC1 activity. The change from a Ca 2+-bound to a Ca 2+-free form of GCAP1 increased susceptibility of Ca 2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca 2+-bound form, only the N-terminus (myristoylated Gly 2-Lys 9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca 2+-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 Ca 2+-dependent inactivation of GCAP1. GCAP1(E 75 D,E 111 D,E 155 D) mutant did not bind Ca 2+ and stimulated GC1 in a [Ca 2+ ]-independent manner. GCAP1 and GCAP2, but not S-100 , a high [Ca 2+ ] free activator of GC1, competed with the triple mutant at high [Ca 2+ ] free , inhibiting GC1 with similar IC 50 's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca 2+ 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. Calcium ions, Ca 2+ , play a crucial role in cellular signaling. Because they are nondegradable, several systems have evolved to regulate the cellular concentration of free Ca 2+ ([Ca 2+ ] free), including intracellular compartmentalization/ sequestration, pumping to the extracellular space, and buffering by Ca 2+-binding proteins. Some of these Ca 2+-binding proteins are also poised to take advantage of transient changes in [Ca 2+ ] free to affect properties of regulatory enzymes and ion channels. In cells that lower their internal [Ca 2+ ] free upon excitation, such as rod and cone photoreceptor cells, distinct types of proteins have evolved that act as activators of effector enzymes when they are in the Ca 2+free state. Guanylate cyclase-activating proteins, GCAP1 1 and GCAP2, were found to fulfill such functions in the regulation of photoreceptor guanylate cyclase (GC1) (1-4). GCAP1 and GCAP2 are acidic, ∼23-kDa, homologous proteins that contain three functional high-affinity, EF-hand Ca 2+-chelating motifs (reviewed in ref 5). At low [Ca 2+ ] free , GCAPs increase the activity of GC1 (6) at least 10-fold (1) by an unknown mechanism. GCAP1 forms a stable complex with GC1, independent of [Ca 2+ ] free. The GC1/GCAP1 complex may switch between two conformations, active and inactive, with the binding or dissociation of Ca 2+ (2, 7). GCAP2 may translocate from the cytosol to the membranebound cyclase when it is free of Ca 2+ and stimulates GC1 activity (8). The N-terminal fatty acid-acylated regions of both GCAPs show weak sequence conservation, and the function of this region remains speculative. It is possible that the N-terminus is flexible and exposed, providing hydrophobic tethering to the membranes for the most efficient stimulation of GC1, as proposed for GCAP1 by Otto-Bruc et al. (9), but this modification is functionally unrelated in the GC1 stimulation by GCAP2, as proposed by Olshevskaya et al. (8).
Biochemistry, 2005
The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca 2+-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca 2+-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca 2+-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca 2+ signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.
Journal of Neurochemistry, 2007
Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca 2+-binding proteins named GC-activating proteins that operate as Ca 2+ sensors and enable cyclases to respond to changes of intracellular Ca 2+ after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca 2+ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca 2+ sensitivity of the Ca 2+ sensor is controlled by the mode of target-sensor interaction.
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...
A Second Calcium Regulator of Rod Outer Segment Membrane Guanylate Cyclase, ROS-GC1: Neurocalcin
Biochemistry, 1999
ROS-GC represents a membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca 2+ signals into the production of the second messenger cyclic GMP. An intriguing feature of the first subfamily member, ROS-GC1, is that it is both stimulated and inhibited by these signals. The inhibitory signals are processed by the cyclase activating proteins, GCAPs. The only known stimulatory signal is by the Ca 2+ -dependent guanylate cyclase activating protein, CD-GCAP. There are two GCAPs, 1 and 2, which link the cyclase with phototransduction, and one CD-GCAP, which is predicted to link ROS-GC1 with its retinal synaptic activity. Individual switches for these GCAPs and CD-GCAP have been respectively defined as CRM1, CRM3, and CRM2. This report defines the identity of a new ROS-GC1 regulator: neurocalcin. A surprising feature of the regulator is that it structurally is a GCAP but functionally behaves as a CD-GCAP. Recombinant neurocalcin stimulates ROS-GC1 in a dose-dependent fashion; the stimulation is Ca 2+ -dependent with an EC 50 of 20 µM; and the modulated domain resides at the C-terminal segment, between amino acids 731 and 1054. Previously, the residence of CRM2 has also been defined in this segment of the cyclase. However, the present study shows that the neurocalcin-regulated domain is distinct from CRM2. This is now designated as CRM4. Thus, the signal transduction mechanisms of neurocalcin and CD-GCAP are different, occurring through different modules of ROS-GC1. Neurocalcin signaling of ROS-GC1 is highly specific. It does not influence the activity of its second subfamily member, ROS-GC2, and of the other retinal guanylate cyclase, atrial natriuretic factor-receptor guanylate cyclase. In conclusion, the findings extend the concept of ROS-GC1's sensing diverse Ca 2+ signals, reveal the identity of its unexpected new Ca 2+ regulator, and show that the regulator acts through its specific cyclase domain. This represents an additional transduction mechanism of Ca 2+ signaling via ROS-GC1.
Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization
Journal of Biological Chemistry, 1999
Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca 2؉ ] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca 2؉ ]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ϳ10 M [Ca 2؉ ] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca 2؉ ] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (ϳ110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca 2؉ -binding proteins used. We also found that [Ca 2؉ ] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.
Biochemistry, 2003
Rod outer segment membrane guanylate cyclase (ROS-GC) transduction system is a central component of the Ca 2+ -sensitive phototransduction machinery. The system is composed of two parts: Ca 2+ sensor guanylate cyclase activating protein (GCAP) and ROS-GC. GCAP senses Ca 2+ impulses and inhibits the cyclase. This operational feature of the cyclase is considered to be unique and exclusive in the phototransduction machinery. A combination of reconstitution, peptide competition, cross-linking, and immunocytochemical studies has been used in this study to show that the GCAP1/ROS-GC1 transduction system also exists in the photoreceptor synaptic (presynaptic) termini. Thus, the presence of this system and its linkage is not unique to the phototransduction machinery. A recent study has demonstrated that the photoreceptor-bipolar synaptic region also contains a Ca 2+ -stimulated ROS-GC1 transduction system ) EMBO J. 21, 2547-2556. In this case, S100 senses Ca 2+ and stimulates the cyclase. The inhibitory and stimulatory Ca 2+ -modulated ROS-GC1 sites are distinct. These findings allow the formation of a new topographic model of ROS-GC1 transduction. In this model, the catalytic module of ROS-GC1 at its opposite ends is flanked by GCAP1 and S100 modules. GCAP1 senses the Ca 2+ impulse and inhibits the catalytic module; S100 senses the impulse and stimulates the catalytic module. Thus, ROS-GC1 acts as a bimodal Ca 2+ signal transduction switch in the photoreceptor bipolar synapse.
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.
Journal of Biological Chemistry, 2001
Guanylyl cyclase-activating proteins are EF-hand Ca 2+ -binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca 2+ binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N′-dimethyl-N-(iodoacetyl)-N′-(7nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys 18 , Cys 29 , Cys 106 , and Cys 125 were observed as a function of Ca 2+ concentration. No Ca 2+ -dependent oligomerization of GCAP1 was observed at physiologically relevant Ca 2+ concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca 2+ dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.