Protein phosphatase 2A dephosphorylates CaBP4 and regulates CaBP4 function.

PURPOSE: CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation. METHODS: The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents. RESULTS: PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells. CONCLUSIONS: This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.

Insight into the role of Ca2+-binding protein 5 in vesicle exocytosis.

PURPOSE: CaBP5 is a neuronal calmodulin-like Ca(2+)-binding protein that is expressed in the retina and in the cochlea. Although CaBP5 knockout mice displayed reduced sensitivity of retinal ganglion cell light responses, the function of CaBP5 in vivo is still unknown. To gain further insight into CaBP5 function, the authors screened for CaBP5-interacting partners. METHODS: Potential retinal interacting partners for CaBP5 were identified using affinity chromatography followed by mass spectrometry and by yeast two-hybrid screening of a bovine retina cDNA library. Interacting partners were further analyzed using coimmunoprecipitation. Immunohistochemistry and subcellular fractionation were performed to determine their colocalization in the retina. The effect of CaBP5 on dopamine release and neurite outgrowth of PC12 cells was analyzed using ELISA and fluorescent labeling. RESULTS: Using affinity chromatography, the authors identified Munc18-1 and myosin VI as interacting partners for CaBP5. Munc18-1 was also identified using the yeast two-hybrid system. Colocalization and coimmunoprecipitation of CaBP5 with these two proteins in retinal tissue further established their physiological interactions. Furthermore, CaBP5 expression in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis. CONCLUSIONS: This study shows that CaBP5 interacts with Munc18-1 and myosin VI, two proteins involved in the synaptic vesicle cycle. Together with the effect of CaBP5 in stimulating neurite outgrowth and vesicle exocytosis in PC12 cells, these results suggest that CaBP5 plays a role in neurotransmitter release.

A targeted proteomics-based pipeline for verification of biomarkers in plasma.

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

DOCK2 is a microglial specific regulator of central nervous system innate immunity found in normal and Alzheimer's disease brain.

Neuroinflammation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Strong epidemiological and experimental evidence supports the use of nonsteroidal anti-inflammatory drugs to reduce AD risk. However, poor outcome in clinical trials and toxicity in a prevention trial have shifted focus away from these cyclooxygenase (COX) inhibitors to seek additional therapeutic targets in the prostaglandin pathway. Previously, the prostaglandin E2 receptor, EP2, was shown to regulate neuroinflammation and reduce Abeta plaque burden in transgenic mice. Unfortunately, widespread EP2 distribution and a direct effect on COX2 induction make EP2 a less desirable target. In this study, we link dedicator of cytokinesis 2 (DOCK2) to the prostaglandin pathway in the brain. Additionally, we show that DOCK2 regulates microglial innate immunity independent of COX2 induction and that DOCK2+ microglia are associated with human AD pathology. Together, these results suggest DOCK2 as a COX2 expression-independent therapeutic target for neurodegenerative diseases such as AD.

Cerebrospinal fluid concentration of brain-derived neurotrophic factor and cognitive function in non-demented subjects.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is an activity-dependent secreted protein that is critical to organization of neuronal networks and synaptic plasticity, especially in the hippocampus. We tested hypothesis that reduced CSF BDNF is associated with age-related cognitive decline. METHODOLOGY/PRINCIPAL FINDINGS, AND CONCLUSIONS/SIGNIFICANCE: CSF concentration of BDNF, Abeta(42) and total tau were measured in 128 cognitively normal adults (Normals), 21 patients with Alzheimer's disease (AD), and nine patients with Mild Cognitive Impairment. Apolipoprotein E and BDNF SNP rs6265 genotype were determined. Neuropsychological tests were performed at baseline for all subjects and at follow-up visits in 50 Normals. CSF BDNF level was lower in AD patients compared to age-matched Normals (p = 0.02). CSF BDNF concentration decreased with age among Normals and was higher in women than men (both p<0.001). After adjusting for age, gender, education, CSF Abeta(42) and total tau, and APOE and BDNF genotypes, lower CSF BDNF concentration was associated poorer immediate and delayed recall at baseline (both p<0.05) and in follow up of approximately 3 years duration (both p<0.01). CONCLUSIONS/SIGNIFICANCE: Reduced CSF BDNF was associated with age-related cognitive decline, suggesting a potential mechanism that may contribute in part to cognitive decline in older individuals.

Quantitation and mapping of cerebral detergent-insoluble proteins in the elderly.

Accumulation of abnormal protein aggregates, detergent-insoluble (DI) proteins and amyloid in the brain are shared features of many neurodegenerative diseases. Previous studies correlating DI proteins and cognitive performance are limited. We addressed these limitations using two sets of autopsy brains, one selected from our Alzheimer's Disease Research Center and the other an unselected series from Adult Changes in Thought (ACT), a population-based study of brain aging. We observed concentrations of 11 proteins and 6 protein variants that can be grouped into three highly correlated clusters: amyloid (A)beta, tau and alpha-synuclein (alpha-syn). While abnormal proteins from each cluster independently correlated with cognitive performance in ACT participants, only increased soluble Abeta oligomers in temporal cortex and increased DI Abeta 42 and DI alpha-syn in prefrontal cortex were negatively correlated with cognitive performance. These data underscore the therapeutic imperative to suppress processes leading to accumulation of soluble Abeta oligomers, DI Abeta 42 and DI alpha-syn, highlight an at least partially independent contribution to cognitive impairment and raise the possibility that the priority for therapeutic targets may vary by brain region in a typical elderly US population.

CSF multianalyte profile distinguishes Alzheimer and Parkinson diseases.

The therapeutic imperative for Alzheimer disease (AD) and Parkinson disease (PD) calls for discovery and validation of biomarkers. Increased cerebrospinal fluid (CSF) tau and decreased amyloid (A) beta42 have been validated as biomarkers of AD. In contrast, there is no validated CSF biomarker for PD. We validated our proteomics-discovered multianalyte profile (MAP) in CSF from 95 control subjects, 48 patients with probable AD, and 40 patients with probable PD. An optimal 8-member MAP agreed with expert diagnosis for 90 control subjects (95%), 36 patients with probable AD (75%), and 38 patients with probable PD (95%). This MAP consisted of the following (in decreasing order of contribution): tau, brain-derived neurotrophic factor, interleukin 8, Abeta42, beta2-microglobulin, vitamin D binding protein, apolipoprotein (apo) AII, and apoE. This first large-scale validation of a proteomic-discovered MAP suggests a panel of 8 CSF proteins that are highly effective at identifying PD and moderately effective at identifying AD.

Quantitative proteomics identifies surfactant-resistant alpha-synuclein in cerebral cortex of Parkinsonism-dementia complex of Guam but not Alzheimer's disease or progressive supranuclear palsy.

Parkinsonism-dementia complex (PDC) remains a significant health burden to the Chamorro population. We tested the hypothesis that quantitative proteomics might provide fresh insight into this enigmatic illness by analyzing proteins resistant to surfactant extraction from patients with Alzheimer's disease (AD) or PDC and their matched controls using isobaric tags for relative and absolute quantification. In addition to the expected increase in abnormal frontal cortical Abeta peptides, tau, ubiquitin, and apolipoprotein E in AD, and tau in PDC, we identified alpha-synuclein (SNCA) as a major abnormal protein in PDC but not AD. We confirmed our isobaric tags for relative and absolute quantification findings by enzyme-linked immunosorbent assay in frontal and temporal cortices. We extended our assays to include a limited number of cases of progressive supranuclear palsy (PSP) and dementia with Lewy bodies; we observed increased abnormal tau but not SNCA in PSP, and abnormal SNCA in dementia with Lewy bodies that was quantitatively similar to PDC. Finally, soluble Abeta oligomers were selectively increased in AD but not PDC or PSP. These results show that frontal and temporal cortex in PDC is distinguished from AD and PSP by its accumulation of abnormal SNCA and suggest that PDC be considered a synucleinopathy as well as a tauopathy.

Apolipoprotein E isoform-dependent dendritic recovery of hippocampal neurons following activation of innate immunity.

BACKGROUND: Innate immune activation, including a role for cluster of differentiation 14/toll-like receptor 4 co-receptors (CD14/TLR-4) co-receptors, has been implicated in paracrine damage to neurons in several neurodegenerative diseases that also display stratification of risk or clinical outcome with the common alleles of the apolipoprotein E gene (APOE): APOE2, APOE3, and APOE4. Previously, we have shown that specific stimulation of CD14/TLR-4 with lipopolysaccharide (LPS) leads to greatest innate immune response by primary microglial cultures from targeted replacement (TR) APOE4 mice and greatest p38MAPK-dependent paracrine damage to neurons in mixed primary cultures and hippocampal slice cultures derived from TR APOE4 mice. In contrast, TR APOE2 astrocytes had the highest NF-kappaB activity and no neurotoxicity. Here we tested the hypothesis that direct activation of CD14/TLR-4 in vivo would yield different amounts of paracrine damage to hippocampal sector CA1 pyramidal neurons in TR APOE mice. METHODS: We measured in vivo changes in dendrite length in hippocampal CA1 neurons using Golgi staining and determined hippocampal apoE levels by Western blot. Neurite outgrowth of cultured primary neurons in response to astrocyte conditioned medium was assessed by measuring neuron length and branch number. RESULTS: Our results showed that TR APOE4 mice had slightly but significantly shorter dendrites at 6 weeks of age. Following exposure to intracerebroventricular LPS, there was comparable loss of dendrite length at 24 hr among the three TR APOE mice. Recovery of dendrite length over the next 48 hr was greater in TR APOE2 than TR APOE3 mice, while TR APOE4 mice had failure of dendrite regeneration. Cell culture experiments indicated that the enhanced neurotrophic effect of TR APOE2 was LDL related protein-dependent. CONCLUSION: The data indicate that the environment within TR APOE2 mouse hippocampus was most supportive of dendrite regeneration while that within TR APOE4 hippocampus failed to support dendrite regeneration in this model of reversible paracrine damage to neurons from innate immune activation, and suggest an explanation for the stratification of clinical outcome with APOE seen in several degenerative diseases or brain that are associated with activated innate immune response.

A novel GCAP1 missense mutation (L151F) in a large family with autosomal dominant cone-rod dystrophy (adCORD).

PURPOSE: To elucidate the phenotypic and biochemical characteristics of a novel mutation associated with autosomal dominant cone-rod dystrophy (adCORD). METHODS: Twenty-three family members of a CORD pedigree underwent clinical examinations, including visual acuity tests, standardized full-field ERG, and fundus photography. Genomic DNA was screened for mutations in GCAP1 exons using DNA sequencing and single-strand conformational polymorphism (SSCP) analysis. Function and stability of recombinant GCAP1-L151F were tested as a function of [Ca(2+)], and its structure was probed by molecular dynamics. RESULTS: Affected family members experienced dyschromatopsia, hemeralopia, and reduced visual acuity by the second to third decade of life. Electrophysiology revealed a nonrecordable photopic response with later attenuation of the scotopic response. Affected family members harbored a C-->T transition in exon 4 of the GCAP1 gene, resulting in an L151F missense mutation affecting the EF hand motif 4 (EF4). This change was absent in 11 unaffected family members and in 100 unrelated normal subjects. GCAP1-L151F stimulation of photoreceptor guanylate cyclase was not completely inhibited at high physiological [Ca(2+)], consistent with a lowered affinity for Ca(2+)-binding to EF4. CONCLUSIONS: A novel L151F mutation in the EF4 hand domain of GCAP1 is associated with adCORD. The clinical phenotype is characterized by early cone dysfunction and a progressive loss of rod function. The biochemical phenotype is best described as persistent stimulation of photoreceptor guanylate cyclase, representing a gain of function of mutant GCAP1. Although a conservative substitution, molecular dynamics suggests a significant change in Ca(2+)-binding to EF4 and EF2 and changes in the shape of L151F-GCAP1.

A novel mutation (I143NT) in guanylate cyclase-activating protein 1 (GCAP1) associated with autosomal dominant cone degeneration.

PURPOSE: To identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation. METHODS: The GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+. RESULTS: A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity. CONCLUSIONS: A novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.

Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes).

The guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins of the calmodulin (CaM) gene superfamily that function in the regulation of photoreceptor guanylate cyclases (GCs). In the mammalian retina, two GCAPs (GCAP 1-2) and two transmembrane GCs have been identified as part of a complex regulatory system responsive to fluctuating levels of free Ca(2+). A third GCAP, GCAP3, is expressed in human and zebrafish (Danio rerio) retinas, and a guanylate cyclase-inhibitory protein (GCIP) has been shown to be present in frog cones. To explore the diversity of GCAPs in more detail, we searched the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) genomes for GCAP-related gene sequences (fuGCAPs and zGCAPs, respectively) and found that at least five additional GCAPs (GCAP4-8) are predicted to be present in these species. We identified genomic contigs encoding fuGCAPl-8, fuGCIP, zGCAPl-5, zGCAP7 and zGCIP. We describe cloning, expression and localization of three novel GCAPs present in the zebrafish retina (zGCAP4, zGCAP5, and zGCAP7). The results show that recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca(2+)-dependent manner. RT-PCR with zGCAP specific primers showed specific expression of zGCAPs and zGCIP in the retina, while zGCAPl mRNA is also present in the brain. In situ hybridization with anti-sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. The presence of at least eight GCAP genes suggests an unexpected diversity within this subfamily of Ca(2+)-binding proteins in the teleost retina, and suggests additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4-8 or GCIP genes.

Guanylate cyclase-activating proteins: structure, function, and diversity.

The guanylate cyclase-activating proteins (GCAPs), Ca2+-binding proteins of the calmodulin gene superfamily, function as regulators of photoreceptor guanylate cyclases. In contrast to calmodulin, which is active in the Ca2+-bound form, GCAPs stimulate GCs in the [Ca2+]-free form and inhibit GCs upon Ca2+ binding. In vertebrate retinas, at least two GCAP1 and two GCs are present, a third GCAP3 is expressed in humans and fish, and at least five additional GCAP4-8 genes have been identified or are predicted in zebrafish and pufferfish. Missense mutations in GCAP1 (Y99C, I143NT, E155G, and P50L) have been associated with autosomal dominant cone dystrophy. Absence of GCAP1/2 in mice delays recovery of the photoresponse, a phenotype consistent with delay in cGMP synthesis. In the absence of GCAP2, GCAP1 supports the generation of wild-type flash responses in both rod and cone cells. Recent progress revealed an unexpected complexity of the GC-GCAP system, pointing, out a number of unsolved questions.

Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475.

Inactivation of the visual G-protein transducin by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP) RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light- and Ca(2+)-sensitive phosphorylation on Ser(475). Because there are much higher RGS9-1 levels in cones than in rods, we investigated whether Ser(475) is phosphorylated in rods using Coneless mice and found that both the phosphorylation and its regulation by light occur in rods. Therefore, we used rod outer segments as the starting material for the purification of RGS9-1 kinase activity. Two major peaks of activity corresponded to protein kinase C (PKC) isozymes, PKCalpha and PKCtheta. A synthetic peptide corresponding to the Ser(475) RGS9-1 sequence and RGS9-1 were substrates for recombinant PKCalpha and PKCtheta. This phosphorylation was removed efficiently by protein phosphatase 2A, an endogenous phosphatase in rod outer segments, but not by PP1 or PP2B. Phosphorylation of RGS9-1 by PKC had little effect on its activity in solution but significantly decreased its affinity for its membrane anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKCtheta immunostaining was at higher levels in cone outer segments than in rod outer segments, as was found for the components of the RGS9-1 GAP complex. Thus, PKC-mediated phosphorylation of RGS9-1 represents a potential mechanism for feedback control of the kinetics of photoresponse recovery in both rods and cones, with this mechanism probably especially important in cones.

Photoreceptor guanylate cyclase variants: cGMP production under control.

Changes in the Ca2+ concentration are thought to affect many processes, including signal transduction in a vast number of biological systems. However, only in few cases the molecular mechanisms by which Ca2+ mediates its action are as well understood as in phototransduction. In dark-adapted photoreceptor cells, the equilibrium level of cGMP is maintained by two opposing activities, such as phosphodiesterase (PDE) and guanylate cyclase (GC). Upon absorption of photons, rhodopsin-G-protein-mediated activation of PDE leads to a transient decrease in [cGMP] and subsequently to lowering of [Ca2+]. In turn, lower [Ca2+] increases net production of cGMP by stimulation of GC until dark conditions are re-established. This activation of GC is mediated by Ca2+ -free forms of Ca2+ -binding proteins termed GC-activating proteins (GCAPs). The last decade brought the molecular identification of GCs and GCAPs in the visual system. Recent efforts have been directed toward understanding the properties of GC at the physiological and structural levels. Here, we summarize the recent progress and present a list of topics of ongoing research.

GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice.

Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca(2+) and cGMP. Ca(2+) regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.

Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebrafish to man.

Calmodulin-like neuronal Ca2+-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca2+-binding motifs. The guanylate cyclase-activating proteins 1-3 (GCAP1-3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1-2 (GC1-2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class-specific residues for the NCBP subfamilies.

Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.

Soluble fusion proteins between single transmembrane photoreceptor guanylyl cyclases and their activators.

Among single-spanning transmembrane receptors (sTMRs), two guanylyl cyclase receptors, GC1 and GC2, are critically important during phototransduction in vertebrate retinal photoreceptor cells. Ca(2+)-free forms of guanylyl cyclase-activating proteins (GCAPs) stimulate GCs intracellularly by a molecular mechanism that is not fully understood. To gain further insight into the mechanism of activation and specificity among these proteins, for the first time, several soluble and active truncated GCs and fusion proteins between intracellular domains of GCs and full-length GCAPs were generated. The GC activity of myristoylated GCAP--(437-1054)GC displayed typical [Ca(2+)] dependence, and was further enhanced by ATP and inhibited by guanylyl cyclase inhibitor protein (GCIP). The myristoyl group of GCAP1 appeared to be critical for the inhibition of GCs at high [Ca(2+)], even without membranes. In contrast, calmodulin (CaM)--(437-1054)GC1 fusion protein was inactive, but could be stimulated by exogenous GCAP1. In a series of experiments, we showed that the activation of GCs by linked GCAPs involved intra- and intermolecular mechanisms. The catalytically productive GCAP1--(437-1054)GC1 complex can dissociate, allowing binding and stimulation of the GC1 fusion protein by free GCAP1. This suggests that the intramolecular interactions within the fusion protein have low affinity and are mimicking the native system. We present evidence that the mechanism of GC activation by GCAPs involves a dimeric form of GCs, involves direct interaction between GCs and GCAPs, and does not require membrane components. Thus, fusion proteins may provide an important advance for further structural studies of photoreceptor GCs and other sTMRs with and without different forms of regulatory proteins.