Advances in Mechanical Thrombectomy for Acute Ischemic Stroke Due to Large Vessel Occlusion.

Acute ischemic stroke (AIS) is a significant cause of global morbidity and mortality, with functional implications for quality of life and long-term disability. The limitations of intravenous thrombolytic therapy for the treatment of AIS, especially for emergent large vessel occlusion (ELVO), have paved the way for alternative therapies and the rapidly evolving landscape of endovascular therapy (EVT). Here, we summarize the major landmark trials that have advanced the field largely due to ongoing biomedical engineering device development that have translated into significantly improved clinical outcomes. Our review describes the clinical success of EVT, and current and future trends.

Resonant plasmon enhancement of light emission from CdSe/CdS nanoplatelets on Au nanodisk arrays.

Semiconducting nanoplatelets (NPLs) have attracted great attention due to the superior photophysical properties compared to their quantum dot analogs. Understanding and tuning the optical and electronic properties of NPLs in a plasmonic environment is a new paradigm in the field of optoelectronics. Here, we report on the resonant plasmon enhancement of light emission including Raman scattering and photoluminescence from colloidal CdSe/CdS nanoplatelets deposited on arrays of Au nanodisks fabricated by electron beam lithography. The localized surface plasmon resonance (LSPR) of the Au nanodisk arrays can be tuned by varying the diameter of the disks. In the case of surface-enhanced Raman scattering (SERS), the Raman intensity profile follows a symmetric Gaussian shape matching the LSPR of the Au nanodisk arrays. The surface-enhanced photoluminescence (SEPL) profile of NPLs, however, follows an asymmetric Gaussian distribution highlighting a compromise between the excitation and emission enhancement mechanisms originating from energy transfer and Purcell effects. The SERS and SEPL enhancement factors depend on the nanodisk size and reach maximal values at 75 and 7, respectively, for the sizes, for which the LSPR energy of Au nanodisks coincides with interband transition energies in the semiconductor platelets. Finally, to explain the origin of the resonant enhancement behavior of SERS and SEPL, we apply a numerical simulation to calculate plasmon energies in Au nanodisk arrays and emission spectra from NPLs in such a plasmonic environment.

Resonant tip-enhanced Raman scattering by CdSe nanocrystals on plasmonic substrates.

Tip-enhanced Raman scattering (TERS) has recently emerged as a powerful technique for studying the local properties of low dimensional materials. Being a plasmon driven system, a dramatic enhancement of the TERS sensitivity can be achieved by an appropriate choice of the plasmonic substrate in the so-called gap-mode configuration. Here, we investigate the phonon properties of CdSe nanocrystals (NCs) utilizing gap-mode TERS. Using the Langmuir-Blodgett technique, we homogeneously deposited submonolayers of colloidal CdSe NCs on two different nanostructured plasmonic substrates. Amplified by resonant gap-mode TERS, the scattering by the optical phonon modes of CdSe NCs is markedly enhanced making it possible to observe up to the third overtone of the LO mode reliably. The home-made plasmonic substrates and TERS tips allow the analysis of the TERS images of CdSe phonon modes with nanometer spatial resolution. The CdSe phonon scattering intensity is strongly correlated with the local electromagnetic field distribution across the plasmonic substrates.

One, four or 100 genera? A new classification of the cone snails.

We present a new classification for the genus Conus sensu lato (family Conidae), based on molecular phylogenetic analyses of 329 species. This classification departs from both the traditional classification in only one genus and from a recently proposed shell- and radula-based classification scheme that separates members of this group into five families and 115 genera. Roughly 140 genus-group names are available for Recent cone snails. We propose to place all cone snails within a single family (Conidae) containing four genera-Conus, Conasprella, Profundiconus and Californiconus (with Conus alone encompassing about 85% of known species)-based on the clear separation of cone snails into four distinct and well-supported groups/lineages in molecular phylogenetic analyses. Within Conus and Conasprella, we recognize 57 and 11 subgenera, respectively, that represent well-supported subgroupings within these genera, which we interpret as evidence of intrageneric distinctiveness. We allocate the 803 Recent species of Conidae listed as valid in the World Register of Marine Species into these four genera and 71 subgenera, with an estimate of the confidence for placement of species in these taxonomic categories based on whether molecular or radula and/or shell data were used in these determinations. Our proposed classification effectively departs from previous schemes by (1) limiting the number of accepted genera, (2) retaining the majority of species within the genus Conus and (3) assigning members of these genera to species groups/subgenera to enable the effective communication of these groups, all of which we hope will encourage acceptance of this scheme.

Surface- and tip-enhanced resonant Raman scattering from CdSe nanocrystals.

Surface- and tip-enhanced resonant Raman scattering (resonant SERS and TERS) by optical phonons in a monolayer of CdSe quantum dots (QDs) is demonstrated. The SERS enhancement was achieved by employing plasmonically active substrates consisting of gold arrays with varying nanocluster diameters prepared by electron-beam lithography. The magnitude of the SERS enhancement depends on the localized surface plasmon resonance (LSPR) energy, which is determined by the structural parameters. The LSPR positions as a function of nanocluster diameter were experimentally determined from spectroscopic micro-ellipsometry, and compared to numerical simulations showing good qualitative agreement. The monolayer of CdSe QDs was deposited by the Langmuir-Blodgett-based technique on the SERS substrates. By tuning the excitation energy close to the band gap of the CdSe QDs and to the LSPR energy, resonant SERS by longitudinal optical (LO) phonons of CdSe QDs was realized. A SERS enhancement factor of 2 × 10(3) was achieved. This allowed the detection of higher order LO modes of CdSe QDs, evidencing the high crystalline quality of QDs. The dependence of LO phonon mode intensity on the size of Au nanoclusters reveals a resonant character, suggesting that the electromagnetic mechanism of the SERS enhancement is dominant. Finally, the resonant TERS spectrum from CdSe QDs was obtained using electrochemically etched gold tips providing an enhancement on the order of 10(4). This is an important step towards the detection of the phonon spectrum from a single QD.

Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea).

We present a large-scale molecular phylogeny that includes 320 of the 761 recognized valid species of the cone snails (Conus), one of the most diverse groups of marine molluscs, based on three mitochondrial genes (COI, 16S rDNA and 12S rDNA). This is the first phylogeny of the taxon to employ concatenated sequences of several genes, and it includes more than twice as many species as the last published molecular phylogeny of the entire group nearly a decade ago. Most of the numerous molecular phylogenies published during the last 15years are limited to rather small fractions of its species diversity. Bayesian and maximum likelihood analyses are mostly congruent and confirm the presence of three previously reported highly divergent lineages among cone snails, and one identified here using molecular data. About 85% of the species cluster in the single Large Major Clade; the others are divided between the Small Major Clade (∼12%), the Conus californicus lineage (one species), and a newly defined clade (∼3%). We also define several subclades within the Large and Small major clades, but most of their relationships remain poorly supported. To illustrate the usefulness of molecular phylogenies in addressing specific evolutionary questions, we analyse the evolution of the diet, the biogeography and the toxins of cone snails. All cone snails whose feeding biology is known inject venom into large prey animals and swallow them whole. Predation on polychaete worms is inferred as the ancestral state, and diet shifts to molluscs and fishes occurred rarely. The ancestor of cone snails probably originated from the Indo-Pacific; rather few colonisations of other biogeographic provinces have probably occurred. A new classification of the Conidae, based on the molecular phylogeny, is published in an accompanying paper.

Horizontal Lloyd mirror patterns from straight and curved nonlinear internal waves.

Experimental observations and theoretical studies show that nonlinear internal waves occur widely in shallow water and cause acoustic propagation effects including ducting and mode coupling. Horizontal ducting results when acoustic modes travel between internal wave fronts that form waveguide boundaries. For small grazing angles between a mode trajectory and a front, an interference pattern may arise that is a horizontal Lloyd mirror pattern. An analytic description for this feature is provided along with comparisons between results from the formulated model predicting a horizontal Lloyd mirror pattern and an adiabatic mode parabolic equation. Different waveguide models are considered, including boxcar and jump sound speed profiles where change in sound speed is assumed 12 m/s. Modifications to the model are made to include multiple and moving fronts. The focus of this analysis is on different front locations relative to the source as well as on the number of fronts and their curvatures and speeds. Curvature influences mode incidence angles and thereby changes the interference patterns. For sources oriented so that the front appears concave, the areas with interference patterns shrink as curvature increases, while convexly oriented fronts cause patterns to expand.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails.

Venoms of predatory marine gastropods of the genus Conus show amazing levels of interspecific diversity and are comprised of a cocktail of peptide neurotoxins, termed conotoxins, that are encoded by large gene families. Conotoxin gene family evolution is characterized by gene duplications and high rates of nonsynonymous substitution among paralogues; yet, what controls the differentiation of venoms among species is not clear. We compared four-loop conotoxin transcripts of six closely related Conus species to examine conotoxin expression patterns among species. The species examined appear to express different numbers of four-loop conotoxin loci and similarity in expression patterns does not seem to correspond with phylogenetic affinity. Moreover, several loci appear to have been independently silenced while others appear to have been revived from previously silenced states. Some loci also appear to exhibit coordinated expression patterns. These results suggest that the evolution of conotoxin expression patterns is incredibly dynamic and the differentiation of venoms of Conus is controlled in part by the evolution of unique conotoxin expression patterns.

Evolution of ecological specialization and venom of a predatory marine gastropod.

Understanding the evolution of ecological specialization is important for making inferences about the origins of biodiversity. Members of the predatory, marine gastropod genus Conus exhibit a variety of diets and the ability to capture prey is linked to a venom comprised of peptide neurotoxins, termed conotoxins. We identified conotoxin transcripts from Conus leopardus, a species of Conus that uniquely preys exclusively on hemichordates, and compared its venom duct transcriptome to that of four other Conus species to determine whether a shift to a specialized diet is associated with changes in the venom composition of this species. We also examined the secondary structure of predicted amino acid sequences of conotoxin transcripts of C. leopardus to identify substitutions that may be linked to specialization on hemichordates. We identified seven distinct conotoxin sequences from C. leopardus that appear to represent transcripts of seven distinct loci. Expression levels and the diversity of conotoxins expressed by C. leopardus are considerably less than those of other Conus. Moreover, gene products of two transcripts exhibited unique secondary structures that have not been previously observed from other Conus. These results suggest that transition to a specialist diet is associated with reduction in the number of components expressed in venoms of Conus and that diverse venoms of Conus are maintained in species with a broad dietary width.

A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium.

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.

Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism.

The atrial natriuretic factor (ANF) signal transduction mechanism consists of the transformation of the signal information into the production of cyclic GMP. The binding of ANF to its receptor, which is also a guanylate cyclase, generates the signal. This cyclase has been termed atrial natriuretic factor receptor guanylate cyclase, ANF-RGC. ANF-RGC is a single transmembrane-spanning protein. The ANF receptor domain resides in the extracellular region of the protein, and the catalytic domain is located in the intracellular region at the C-terminus of the protein. Thus, the signal is relayed progressively from the receptor domain to the catalytic domain, where it is converted into the formation of cyclic GMP. The first transduction step is the direct binding of ATP with ANF-RGC. This causes allosteric regulation of the enzyme and primes it for the activation of its catalytic moiety. The partial structural motif of the ATP binding domain in ANF-RGC has been elucidated, and it has been named ATP regulatory module (ARM). In this presentation, we provide a brief review of the ATP-regulated transduction mechanism and the ARM model. The model depicts a configuration of the ATP-binding pocket that has been experimentally validated, and the model shows that the ATP-dependent transduction process is a two- (or more) step event. The first step involves the binding of ATP with its ARM. This partially activates the cyclase and prepares it for the subsequent steps, which are consistent with its being phosphorylated and attaining the fully activated state.

Negatively calcium-modulated membrane guanylate cyclase signaling system in the rat olfactory bulb.

The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.

Three dimensional atomic model and experimental validation for the ATP-Regulated Module (ARM) of the atrial natriuretic factor receptor guanylate cyclase.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Impairment of the rod outer segment membrane guanylate cyclase dimerization in a cone-rod dystrophy results in defective calcium signaling.

Rod outer segment membrane guanylate cyclase1 (ROS-GC1) is the original member of the membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals in the sensory neurons. In the vertebrate retinal neurons, ROS-GC1 is pivotal for the operations of phototransduction and, most likely, of the synaptic activity. The phototransduction- and the synapse-linked domains are separate, and they are located in the intracellular region of ROS-GC1. These domains sense Ca(2+) signals via Ca(2+)-binding proteins. These proteins are ROS-GC activating proteins, GCAPs. GCAPs control ROS-GC1 activity through two opposing regulatory modes. In one mode, at nanomolar concentrations of Ca(2+), the GCAPs activate the cyclase and as the Ca(2+) concentrations rise, the cyclase is progressively inhibited. This mode operates in phototransduction via two GCAPs: 1 and 2. The second mode occurs at micromolar concentrations of Ca(2+) via S100beta. Here, the rise of Ca(2+) concentrations progressively stimulates the enzyme. This mode is linked with the retinal synaptic activity. In both modes, the final step in Ca(2+) signal transduction involves ROS-GC dimerization, which causes the cyclase activation. The identity of the dimerization domain is not known. A heterozygous, triple mutation -E786D, R787C, T788M- in ROS-GC1 has been connected with autosomal cone-rod dystrophy in a British family. The present study shows the biochemical consequences of this mutation on the phototransduction- and the synapse-linked components of the cyclase. (1) It severely damages the intrinsic cyclase activity. (2) It significantly raises the GCAP1- and GCAP2-dependent maximal velocity of the cyclase, but this compensation, however, is not sufficient to override the basal cyclase activity. (3) It converts the cyclase into a form that only marginally responds to S100beta. The mutant produces insufficient amounts of the cyclic GMP needed to drive the machinery of phototransduction and of the retinal synapse at an optimum level. The underlying cause of the breakdown of both types of machinery is that, in contrast to the native ROS-GC1, the mutant cyclase is unable to change from its monomeric to the dimeric form, the form required for the functional integrity of the enzyme. The study defines the CORD in molecular terms, at a most basic level identifies a region that is critical in its dimer formation, and, thus, discloses a single unifying mechanistic theme underlying the complex pathology of the disease.

Evolutionary diversification of multigene families: allelic selection of toxins in predatory cone snails.

In order to investigate the evolution of conotoxin multigene families among two closely related vermivorous CONUS: species, we sequenced 104 four-loop conotoxin mRNAs from two individuals of CONUS: ebraeus and compared these with sequences already obtained from CONUS: abbreviatus. In contrast to the diversity of conotoxin sequences obtained from C. abbreviatus, only two common sequence variants were recovered from C. ebraeus. Segregation patterns of the variants in these two individuals and restriction digests of four-loop conotoxin amplification products from nine additional individuals suggest that the common variants are alleles from a single locus. These two putative alleles differ at nine positions that occur nonrandomly in the toxin-coding region of the sequences. Moreover, all substitutions are at nonsynonymous sites and are responsible for seven amino acid differences among the predicted amino acid sequences of the alleles. These results imply that conotoxin diversity is driven by strong diversifying selection and some form of frequency-dependent or overdominant selection at conotoxin loci, and they suggest that diverse conotoxin multigene families can originate from duplications at polymorphic loci. Furthermore, none of the sequences recovered from C. ebraeus appeared to be orthologs of loci from C. abbreviatus, and attempts to amplify orthologous sequences with locus-specific primers were unsuccessful among these species. These patterns suggest that venoms of closely related CONUS: species may differ due to the differential expression of conotoxin loci.

Rod outer segment membrane guanylate cyclase type 1-linked stimulatory and inhibitory calcium signaling systems in the pineal gland: biochemical, molecular, and immunohistochemical evidence.

Recent evidence indicates the presence of a novel alpha(2D/A)-adrenergic receptor (alpha(2D/A)-AR) linked membrane guanylate cyclase signal transduction system in the pineal gland. This system operates via a Ca(2+)-driven rod outer segment membrane guanylate cyclase (ROS-GC). In the present study, this transduction system has been characterized via molecular, immunohistochemical, and biochemical approaches. The two main components of the system are ROS-GC1 and its Ca(2+) regulator, S100B. Both components coexist in pinealocytes where the signaling component alpha(2D/A)-AR also resides. The presence of ROS-GC2 was not detected in the pineal gland. Thus, transduction components involved in processing alpha(2D/A)-AR-mediated signals are Ca(2+), S100B, and ROS-GC1. During this investigation, an intriguing observation was made. In certain pinealocytes, ROS-GC1 coexisted with its other Ca(2+) modulator, guanylate cyclase activating protein type 1 (GCAP1). In these pinealocytes, S100B was not present. The other GCAP protein, GCAP2, which is also a known modulator of ROS-GC in photoreceptors, was not present in the pineal gland. The results establish the identity of an alpha(2D/A)-AR-linked ROS-GC1 transduction system in pinealocytes. Furthermore, the findings show that ROS-GC1, in a separate subpopulation of pinealocytes, is associated with an opposite Ca(2+) signaling pathway, which is similar to phototransduction in retina. Thus, like photoreceptors, pinealocytes sense both positive and negative Ca(2+) signals, where ROS-GC1 plays a pivotal role; however, unlike photoreceptors, the pinealocyte is devoid of the ROS-GC2/GCAP2 signal transduction system.

Three dimensional atomic model and experimental validation for the ATP-Regulated Module (ARM) of the atrial natriuretic factor receptor guanylate cyclase.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Regions in vertebrate photoreceptor guanylyl cyclase ROS-GC1 involved in Ca(2+)-dependent regulation by guanylyl cyclase-activating protein GCAP-1.

The membrane bound guanylyl cyclase (GC) photoreceptor membrane GC1 (ROS-GCI) of photoreceptor cells synthesizes cGMP, the intracellular transmitter of vertebrate phototransduction. The activity of ROS-GCI is controlled by small Ca(2+)-binding proteins, named GC-activating proteins (GCAPs). We identified and characterized two short regulatory regions (M445-L456 and L503-1522) in the juxtamembrane domain (JMD) of ROS-GC1 by peptide competition and mutagenesis studies. Both regions are critical for the activation of ROS-GCI by GCAP-1.

A second calcium regulator of rod outer segment membrane guanylate cyclase, ROS-GC1: neurocalcin.

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 microM; 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.