Regions in vertebrate photoreceptor guanylyl cyclase ROS-GC1 involved in Ca2+-dependent regulation by guanylyl cyclase-activating protein GCAP-1 (original) (raw)
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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)
Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)
Journal of Biological Chemistry, 2014
Background: GCAP1 regulates cGMP synthesis in photoreceptors in response to light. Results: Mutagenesis of the entire GCAP1 surface reveals its guanylyl cyclase interface. Conclusion: The interface forms a compact patch that enables both primary binding to and allosteric activation of the target enzyme. Significance: Guanylyl cyclase activation by GCAP1 is indispensable for vision and survival of photoreceptors. Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metalbinding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca 2؉ binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94. Retinal membrane guanylyl cyclase (RetGC) 2 and RetGCactivating proteins (GCAPs) play a critical role in the physiol
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...
Ca 2+ -dependent Regulation of Phototransduction
Photochemistry and Photobiology, 2008
Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca 2+ concentration in the photoreceptor cytosol sensed by Ca 2+ -binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca 2+ concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca 2+ -triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca 2+ sensors in the modulation of phototransduction.
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.
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.
Retinal Guanylyl Cyclase-Activating Protein 1 and 2
Encyclopedia of Signaling Molecules, 2016
Historical Background Retinal guanylyl cyclases (RetGCs) in retinal rod and cone photoreceptors are regulated by a family of EF-hand Ca 2+ sensor proteins called guanylyl cyclase-activating proteins (GCAP1-8) that belong to the neuronal calcium sensor (NCS) family. Mammalian GCAPs (GCAP1 and GCAP2) activate RetGCs at low Ca 2+ levels in light-activated photoreceptor cells and inhibit RetGC activity at higher Ca 2+ levels in darkadapted photoreceptors. The Ca 2+-sensitive RetGC activity controlled by GCAPs is an important mechanism of visual recovery and light adaptation of phototransduction. Mutations in either RetGCs or GCAPs that disable this Ca 2+-sensitive cyclase activity are genetically linked to retinal disease. Here I review atomic-level structures of GCAP1 in both Ca 2+-free/Mg 2+-bound (activator) and Ca 2+-saturated (inhibitory) states, as well as the structure of Ca 2+-saturated GCAP2. The structure of GCAP2 reveals an exposed N-terminus that may be important for Ca 2+-dependent membrane anchoring of the myristoyl group. By contrast, the structures of Ca 2+-free and Ca 2+-bound forms of GCAP1 each contain a covalently attached myristoyl group that is sequestered in a hydrophobic protein cavity formed by helices at both the N-and C-terminus. Hence, myristoylated GCAP1 is not targeted to bilayer membranes. The Ca 2+-free activator form of GCAP1 contains Mg 2+ bound at the second EF-hand (EF2) that is essential for activating RetGC. The Ca 2+ saturated form of GCAP1 contains Ca 2+ bound at EF2, EF3, and EF4. Ca 2+-dependent conformational changes are most apparent in EF2 and in the Ca 2+ switch helix (residues 169-174) and will be discussed in terms of a proposed mechanism for Ca 2+-dependent activation of retinal guanylyl cyclases.
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...
Journal of Neuroscience, 2004
Guanylyl cyclase-activating proteins (GCAPs) are Ca 2ϩ -binding proteins that activate guanylyl cyclase when free Ca 2ϩ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca 2ϩ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca 2ϩ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca 2ϩ , the normal resting Ca 2ϩ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca 2ϩ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca 2ϩ -dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca 2ϩ in the outer segment, which may trigger the apoptotic process.
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.