Membrane Guanylate Cyclase Research Articles

Modulation of the cGMP signaling pathway by melatonin in pancreatic β‐cells

Melatonin influences the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) signaling pathway in pancreatic beta-cells via a receptor-mediated mechanism. In the present study, it was determined how the regulation of cGMP concentrations by melatonin proceeds. The results provide evidence that melatonin acts via the soluble guanylate cyclase (sGC), as molecular investigations demonstrated that long-term incubation with melatonin significantly reduced the expression levels of the sGC mRNA in rat insulinoma beta-cells (INS1) cells, whereas mRNA expression of membrane guanylate cyclases was unaffected. Incubation with melatonin abolished the S-nitrosoacetyl penicillamine-induced increase of cGMP concentrations in INS1 cells. In addition, the cGMP-inhibitory effect of melatonin was reversed by preincubation with the sGC inhibitors 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1-one and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one. Nitric oxide (NO) production was not influenced after 1 hr of melatonin application, but was influenced after a 4 hr incubation period. Preincubation of INS1 cells with the NO synthase inhibitor N(G)-monomethyl-l-arginine did not abolish the cGMP-inhibitory effect of melatonin. Transcripts of cyclic nucleotide-gated (CNG) channels were significantly reduced after melatonin treatment in a dose-dependent manner, indicating the involvement of these channels in mediating the melatonin effect in INS1 cells. The results of this study demonstrate that melatonin mediates its inhibitory effect on cGMP concentrations in pancreatic beta-cells by inhibiting the sGC, but does not influence NO concentration or NO synthase activity in short-term incubation experiments. In addition, it was demonstrated that melatonin is involved in modulation of CNG channel mRNA.

Hippocalcin, new Ca2+ sensor of a ROS-GC subfamily member, ONE-GC, membrane guanylate cyclase transduction system

Hippocalcin is a member of the neuronal Ca(2+) sensor protein family. Among its many biochemical functions, its established physiological function is that via neuronal apoptosis inhibitory protein it protects the neurons from Ca(2+)-induced cell death. The precise biochemical mechanism/s, through which hippocalcin functions, is not clear. In the present study, a new mechanism by which it functions is defined. The bovine form of hippocalcin (BovHpca) native to the hippocampus has been purified, sequenced, cloned, and studied. The findings show that there is the evolutionary conservation of its structure. It is a Ca(2+)-sensor of a variant form of the ROS-GC subfamily of membrane guanylate cyclases, ONE-GC. It senses physiological increments of Ca(2+) with a K(1/2) of 0.5 microM and stimulates ONE-GC or ONE-GC-like membrane guanylate cyclase. The Hpca-modulated ONE-GC-like transduction system exists in the hippocampal neurons. And hippocalcin-modulated ONE-GC transduction system exists in the olfactory receptor neuroepithelium. The Hpca-gene knock out studies demonstrate that the portion of this is about 30% of the total membrane guanylate cyclase transduction system. The findings establish Hpca as a new Ca(2+) sensor modulator of the ROS-GC membrane guanylate cyclase transduction subfamily. They support the concept on universality of the presence and operation of the ROS-GC transduction system in the sensory and sensory-linked neurons. They validate that the ROS-GC transduction system exists in multiple forms. And they provide an additional mechanism by which ROS-GC subfamily acts as a transducer of the Ca(2+) signals originating in the neurons.

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM's structural motif, (669)WTAPELL(675). This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the (669)WTAPELL(675) motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.

Structural Characterisation of the E. coli Heat Stable Enterotoxin STh

E. coli heat stable enterotoxin STa is an agonist of the membrane guanylate cyclase C whose endogenous ligands are the peptide hormones guanylin and uroguanylin. Whereas these peptides contain only two disulfide bonds, STa is stabilized by one additional disulfide bridge. We chemically synthesized the enterotoxin STh that originates from the E. coli strain found in humans, and we determined its structure and its dynamics by nuclear magnetic resonance spec- troscopy and molecular dynamics calculations. Chemical synthesis clearly proved successful and resulted in the formation of the native disulfide bonds. The endogenous ligands guanylin and uroguanylin show the same general structural features and dynamics properties as the enterotoxin.

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) calcium-modulated transduction system in the sperm

Evaluation of the presence of a Ca(2+)-regulated membrane guanylate cyclase signal transudation system in the spermatozoa. Experimental study. Research university laboratory. Human sperm obtained from healthy donors who met the criteria of the World Health Organization for normozoospermia and bovine semen collected from bulls of proven fertility. Radioimmunoassay and immunohistochemistry of human and bovine spermatozoa. The membrane guanylate cyclase activity and the presence of membrane guanylate cyclase transduction machinery components in the spermatozoa. The identity of a Ca(2+)-modulated membrane guanylate cyclase transduction machinery in human and bovine spermatozoa has been documented. The machinery is both inhibited and stimulated within nanomolar to semimicromolar range of free Ca(2+). The transduction component of this machinery is the rod outer segment membrane guanylate cyclase type 1 (ROS-GC1). The enzyme coexists with three Ca(2+)-dependent modulators: guanylate cyclase activating protein type 1 (GCAP1), S100B and neurocalcin delta. ROS-GC1 and its modulators are present in the heads and tails of both species' spermatozoa. The coexpression of ROS-GC1 and its activators in spermatozoa suggests that the Ca(2+)-modulated ROS-GC1 transduction system may be a part of the fertilization machinery.

Neurocalcin δ Modulation of ROS-GC1, a New Model of Ca2+ Signaling

ROS-GC1 membrane guanylate cyclase is a Ca(2+) bimodal signal transduction switch. It is turned "off" by a rise in free Ca(2+) from nanomolar to the semicromolar range in the photoreceptor outer segments and the olfactory bulb neurons; by a similar rise in the bipolar and ganglion retinal neurons it is turned "on". These opposite operational modes of the switch are specified by its Ca(2+) sensing devices, respectively termed GCAPs and CD-GCAPs. Neurocalcin delta is a CD-GCAP. In the present study, the neurocalcin delta-modulated site, V(837)-L(858), in ROS-GC1 has been mapped. The location and properties of this site are unique. It resides within the core domain of the catalytic module and does not require the alpha-helical dimerization domain structural element (amino acids 767-811) for activating the catalytic module. Contrary to the current beliefs, the catalytic module is intrinsically active; it is directly regulated by the neurocalcin delta-modulated Ca(2+) signal and is dimeric in nature. A fold recognition based model of the catalytic domain of ROS-GC1 was built, and neurocalcin delta docking simulations were carried out to define the three-dimensional features of the interacting domains of the two molecules. These findings define a new transduction model for the Ca(2+) signaling of ROS-GC1.

ONE-GC membrane guanylate cyclase, a trimodal odorant signal transducer

The Ca2+-modulated ONE-GC membrane guanylate cyclase is a central component of the cyclic GMP signaling in odorant transduction. It is a single transmembrane spanning modular protein. Its intracellular region contains Ca2+ sensor recognition domains linked to GCAP1 and to neurocalcin δ, and a catalytic module. These domains sense increments in free Ca2+ and stimulate the catalytic module. The present study makes three significant mechanistic advancements. First, to date no ligand for the extracellular (ext) domain is known, for this reason ONE-GC has been deemed as an orphan receptor. The present study identifies its ligand. Uroguanylin stimulates ONE-GC through its ext domain. Second, so far no ligand is known that directly stimulates the catalytic module of any membrane guanylate cyclase. The presented evidence shows that in the presence of the semimicromolar range of free Ca2+, neurocalcin binds to the catalytic module and stimulates ONE-GC. Thus, ONE-GC has trimodal regulation, two occurring intracellularly and one extracellularly. Third, guanylin, a urine odorant, does not directly stimulate ONE-GC. This challenges the proposed hypothesis that the guanylin odorant signal occurs via ONE-GC [T. Leinders-Zufall, R.E. Cockerham, S. Michalakis, M. Biel, D.L. Garbers, R.R. Reed, F. Zufall, S.D. Munger, Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium, Proc. Natl. Acad. Sci. USA. 104 (2007) 14507–14512].

Calcium‐Modulated Rod Outer Segment Membrane Guanylate Cyclase Type 1 Transduction Machinery in the Testes

The importance of the second messengers, Ca(2+) and cyclic GMP, for the process of fertilization is well established; the mechanisms for their intracellular regulations in the testes are, however, poorly understood. This study documents the biochemical, molecular, and functional identity of a Ca(2+)-modulated membrane guanylate cyclase transduction machinery in bovine testes. The machinery is both inhibited and stimulated by free Ca(2+) levels. The Ca(2+)-sensor component of the inhibitory mode of the machinery is GCAP1 (guanylate cyclase activating protein type 1) and for the stimulatory mode is S100B. The transduction component is a Ca(2+)-driven rod outer segment membrane guanylate cyclase type 1, ROS-GC1. The cyclase is predominantly expressed in spermatogenic cells. GCAP1 expression is restricted to a small population of spermatogonia, whereas S100B is present in the majority of spermatocytes and spermatids. The expression of GCAP1 and S100B in spermatocytes and spermatids is mutually exclusive.

Interaction of atrial natriuretic peptide, urodilatin, guanylin and uroguanylin in the isolated perfused rat kidney

Escherichia coli heat-stable enterotoxin (STa), guanylin and uroguanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, guanylin and uroguanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced guanylin (0.19 μM) natriuretic activity (%ENa +; from 18.5 ± 4.25 to 31.5 ± 1.69, P < 0.05, 120 min) and its kaliuretic activity (%EK +; from 24.5 ± 4.43 to 50.6 ± 3.84, P < 0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05 ± 3.00 to 37.8 ± 2.95, P < 0.05, 120 min) and kaliuretic (from 33.2 ± 3.52 to 42.83 ± 2.45, P < 0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 μM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 μM) increase in %ENa + (from 35.9 ± 2.37 to 14.8 ± 1.93, P < 0.05, 120 min), and in %EK + (from 51.0 ± 4.43 to 38.8 ± 3.61, P < 0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa + = 31.0 ± 1.93, P < 0.05, 120 min) and kaliuresis (to %EK + = 54.2 ± 3.61, P < 0.05, 120 min), and inhibited the %ENa + of uroguanylin (0.31 μM) to 17.9 ± 1.67 as well as its %EK + to 24.3 ± 3.13 (both at 120 min, P < 0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca(2+) sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca(2+)-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca(2+) myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca(2+) myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca(2+)-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca(2+)-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.

Calcium-modulated ciliary membrane guanylate cyclase transduction machinery: Constitution and operational principles

Odorant transduction is a biochemical process by which the odorant signal generates the electric signal. The cilia of the olfactory neuroepithelium are the sites of this process. This study documents the detailed biochemical, structural and functional description of an odorant-responsive Ca2+-modulated membrane guanylate cyclase transduction machinery in the cilia. Myristoylated (myr)-neurocalcin δ is the Ca2+-sensor component and the cyclase, ONE-GC, the transduction component of the machinery. Myr-neurocalcin δ senses increments in free Ca2+, binds to a defined domain of ONE-GC and stimulates the cyclase. The findings enable the formulation of an odorant transduction model in which three pivotal signaling components—Ca2+, myr-neurocalcin δ and ONE-GC—of the transduction machinery are locked. A glaring feature of the model is that its Ca2+-dependent operational principle is opposite to the phototransduction model. (Mol Cell Biochem 267: 107–122, 2004)

Two membrane juxtaposed signaling modules in ANF-RGC are interlocked

Atrial natriuretic factor (ANF) receptor guanylate cyclase ANF-RGC is a single transmembrane spanning modular protein. Juxtaposed to each side of the transmembrane module is a Cys 423–Cys 432 disulfide ANF signaling module motif and the ATP-regulated transduction module (ARM) motif. The signaling module motif is conserved in nearly all membrane guanylate cyclases and is believed to be critical in the signaling activities of all membrane guanylate cyclases. The present study with the model system of the olfactory membrane guanylate cyclase shows that this concept is not valid. Furthermore, the study shows that in ANF-GC the signaling motif works through the ARM domain. A new signaling model is proposed where in its natural state the disulfide structural motif represses the ARM domain activity, which, in turn, represses the catalytic module activity of ANF-RGC. ANF signaling relieves the disulfide structural motif restraint on the ARM inhibition and stimulates the catalytic module of the cyclase.

The Calcium-Sensor Guanylate Cyclase Activating Protein Type 2 Specific Site in Rod Outer Segment Membrane Guanylate Cyclase Type 1

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.

ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase

ATP is an obligatory agent for the atrial natriuretic factor (ANF) and the type C natriuretic peptide (CNP) signaling of their respective receptor guanylate cyclases, ANF-RGC and CNP-RGC. Through a common mechanism, it binds to a defined ARM domain of the cyclase, activates the cyclase and transduces the signal into generation of the second messenger cyclic GMP. In this presentation, the authors review the ATP-regulated transduction mechanism and refine the previously simulated three-dimensional ARM model (Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2000;214:7–14; reviewed in: Sharma RK, Yadav P, Duda T. Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism. Can J Physiol Pharmacol 2001;79: 682–91; Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3–30). The model depicts the ATP-binding dependent configurational changes in the ARM and supports the concept that in the first step, ATP partially activates the cyclase and primes it for the subsequent transduction steps, resulting in full activation of the cyclase.

Calcium-modulated ciliary membrane guanylate cyclase transduction machinery: Constitution and operational principles

Odorant transduction is a biochemical process by which the odorant signal generates the electric signal. The cilia of the olfactory neuroepithelium are the sites of this process. This study documents the detailed biochemical, structural and functional description of an odorant-responsive Ca2+ -modulated membrane guanylate cyclase transduction machinery in the cilia. Myristoylated (myr)-neurocalcin delta is the Ca2+ -sensor component and the cyclase, ONE-GC, the transduction component of the machinery. Myr-neurocalcin delta senses increments in free Ca2+, binds to a defined domain of ONE-GC and stimulates the cyclase. The findings enable the formulation of an odorant transduction model in which three pivotal signaling components--Ca2+, myr-neurocalcin delta and ONE-GC--of the transduction machinery are locked. A glaring feature of the model is that its Ca2+ -dependent operational principle is opposite to the phototransduction model.

S100B-modulated Ca 2+-dependent ROS-GC1 transduction machinery in the gustatory epithelium: a new mechanism in gustatory transduction

Gustatory transduction is a biochemical process by which the gustatory signal generates the electric signal. The microvilli of the taste cells in the gustatory epithelium are the sites of gustatory transduction. This study documents the biochemical, molecular, and functional identity of the Ca 2+-modulated membrane guanylate cyclase transduction machinery in the bovine gustatory epithelium. The machinery is a two-component system: the Ca 2+-sensor protein, S100B; and the transducer, ROS-GC1. S100B senses increments in free Ca 2+, undergoes conformational change, binds to the domain amino acids (aa) Gly962-Asn981 and via the transduction domain aa Ile1030-Gln1041 activates ROS-GC1, generating the second messenger, cyclic GMP. In a recent study, operational presence of this machinery has been demonstrated in the photoreceptor bipolar synapse [Duda et al., EMBO J. 21 (2002) 2547]. Thus, the machinery has a broader role in sensory perceptions, vision in the retinal neurons and gustation in the tongue. The entry of the ROS-GC transduction machinery defines the beginning of a new paradigm of Ca 2+ signaling in the tongue.

Stimulation of guanylate cyclase in rat parotid membranes by phosphate

The natriuretic peptide receptor guanylate cyclases are members of the membrane-bound guanylate cyclase family. Atrial natriuretic pepticle (ANP) stimulates guanylate cyclase A, which is predominantly expressed in the rat parotid gland. ATP is well known to increase ANP-stimulated guanylate cyclase, with Mg2+ as a cofactor. We investigated the regulation of guanylate cyclase activity in rat parotid membranes with Mn2+ as a cofactor, because enzyme activity was much higher with Mn2+ than with Mg2+. ANP (10(-7)M) stimulated guanylate cyclase activity to 120%-130% of the control level, and ATP (0.1-1.0 mM) depressed its activity, with or without ANP, to 70%-80% of the control. Enzyme activity was increased by the addition of phosphate (5-20 mM). In the presence of phosphate (5 mM), guanylate cyclase with and without ANP was maximally stimulated to 5- and 6.6-fold of the control, respectively. The net stimulation of guanylate cyclase by ANP was increased at ATP concentrations between 0.2 and 0.5 mM in the presence of phosphate (5 mM or 10 mM), but no change was observed in the absence of phosphate. Phosphate not only stimulated guanylate cyclase in the absence of ATP but altered the ATP regulation of ANP-stimulated guanylate cyclase. The stimulation of guanylate cyclase by phosphate may depend on a peculiarity of rat parotid membranes.

Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis.

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.

Structural, Biochemical, and Functional Characterization of the Calcium Sensor Neurocalcin δ in the Inner Retinal Neurons and Its Linkage with the Rod Outer Segment Membrane Guanylate Cyclase Transduction System

This study documents the detailed biochemical, structural, and functional identity of a novel Ca(2+)-modulated membrane guanylate cyclase transduction system in the inner retinal neurons. The guanylate cyclase is the previously characterized ROS-GC1 from the photoreceptor outer segments (PROS), and its new modulator is neurocalcin delta. At the membrane, the myristoylated form of neurocalcin delta senses submicromolar increments in free Ca(2+), binds to its specific ROS-GC1 domain, and stimulates the cyclase. Neurocalcin delta is not present in PROS, indicating the absence of the pathway in the outer segments and the dissociation of its linkage with phototransduction. Thus, the pathway is linked specifically with the visual transduction machinery in the secondary neurons of the retina. With the inclusion of this pathway, the findings broaden the understanding of the existing mechanisms showing how ROS-GC1 is able to receive and transduce diverse Ca(2+) signals into the cell-specific generation of second-messenger cyclic GMP in the retinal neurons.

Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity.

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca(2+)-dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (kcat/Km) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on kcat/Km. The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca(2+)-sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca(2+)], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca(2+)-sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two 'GCAP modes' that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca(2+) in photoreceptor cells.