The Calcium-Sensor Guanylate Cyclase Activating Protein Type 2 Specific Site in Rod Outer Segment Membrane Guanylate Cyclase Type 1 † (original) (raw)
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Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism
Molecular and cellular …, 1998
Ca 2+-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca 2+ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca 2+ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext-(deleted aa 8-408); kin-(deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC 50 of about 140 nM. A previous study has shown that under identical conditions the kinand the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.
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).
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.
Journal of Biological Chemistry, 1999
Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca 2؉-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca 2؉ concentration (<100 nM) and inhibits them at high Ca 2؉ (>500 nM). The light-induced lowering of the Ca 2؉ level from ϳ500 nM in the dark to ϳ50 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 Ca 2؉ 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 Å in comparing the Ca 2؉-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 (Leu 24 , Trp 27 , Phe 31 , Phe 45 , Phe 48 , Phe 49 , Tyr 81 , Val 82 , Leu 85 , and Leu 89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.
The Journal of biological chemistry, 2001
Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both t...
The EMBO Journal, 2002
This study documents the identity of a calciumregulated membrane guanylate cyclase transduction system in the photoreceptor-bipolar synaptic region. The guanylate cyclase is the previously characterized ROS-GC1 from the rod outer segments and its modulator is S100b. S100b senses increments in free Ca 2+ and stimulates the cyclase. Speci®city of photoreceptor guanylate cyclase activation by S100b is validated by the identi®cation of two S100b-regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are speci®c for S100b and not for another regulator of ROS-GC1, guanylate cyclase-activating protein 1. One site comprises amino acids (aa) Gly962±Asn981, the other, aa Ile1030±Gln1041. The former represents the binding site. The latter binds S100b only marginally, yet it is critical for control of maximal cyclase activity. The ®ndings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane-bound guanylate cyclase is regulated by small Ca 2+-sensor proteins.
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...
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.
Biochemical and Biophysical Research Communications, 2007
Guanylate cyclase-activating protein 2 (GCAP2) is expressed in vertebrate photoreceptors cells where it regulates the activity of membrane bound guanylate cyclases in a Ca 2+-dependent manner. The essential trigger step involves a Ca 2+-induced conformational change in GCAP2. We investigated these Ca 2+-dependent changes by probing the cysteine accessibility in wild type and mutant GCAP2 forms with the thiol-modifying reagent 5,5 0-dithio-bis-(2-nitrobenzoic acid) (DTNB). Cysteine residues in position 35 and 111 displayed a restricted accessibility in the presence of Ca 2+ , whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca 2+. Our data indicate that the Ca 2+-sensitivity of GCAP2 is significantly controlled by its third Ca 2+-binding site, EF-hand 3.