Estudio del Fenómeno Social Media en las grandes corporaciones del sector TIC

Este trabajo trata el estudio de la implementación y desarrollo de las diversas plataformas del social media: redes sociales y del conocimiento, blogs y herramientas colaborativas en el ámbito corporativo de la empresa. El estudio recopila información de consultoras tecnológicas, de artículos y de diversas plataformas social media y se realiza una investigación sobre el tema planteado. Se incluye el análisis de 42 encuestas a profesionales de dos grandes empresas de las telecomunicaciones en España. Estas dos empresas cuentan una con cerca de 28000 empleados y la otra con más 300 empleados en sus filiales españolas. Ambas tienen una importante presencia internacional. Estas dos empresa se diferencian de otras empresas del sector de las telecomunicaciones en que están apostando en la implementación del social media en sus procesos internos. Además se incluye el estudio y análisis de las estadísticas de uso y de una series de encuestas realizadas en el muro de la red social corporativa de una multinacional de las telecomunicaciones durante tres meses. Se presenta una nueva cultura social de empresa innovadora en áreas como la gestión del conocimiento, comunicación interna, formación e innovación. Y se ofrece una visión cuantitativa y de la implantación del social media en los procesos de una empresa. Se desarrolla una exposición donde se detalla el proceso de estudio de las diferentes plataformas social media y áreas de aplicación en la empresa, el estudio de los aspectos legales de su aplicación y uso y la implementación y desarrollo. Asimismo se expone un análisis teórico-práctico del cálculo del retorno de la inversión (ROI) y por último un análisis de la información recopilada en las encuestas y en el estudio estadístico de la red social corporativa. Los datos de las encuestas fueron analizados mediante estadística descriptiva basada en gráficos y tablas de contingencia donde se calculan residuos y porcentajes totales para analizar la dependencia entre el social media, eficiencia, productividad y cuenta de resultados, además del análisis de la aportación del social media a la misión, comunicación interna y gestión del conocimiento en la empresa. También se realizan cálculos de distribuciones Chi-cuadrado para demostrar la dependencia del social media-productividad y del GAP que relaciona la importancia y el nivel de satisfacción del social media. En el análisis teórico-práctico se toman como parámetros los beneficios, costes, flexibilidad y riesgo. Los beneficios van ligados a la productividad, gestión del conocimiento, capital humano y procesos internos. Los costes a las licencias de software, administración, implementación y formación. A partir de estos parámetros se realizó el estudio de un modelo de empresa que representa a una gran empresa de las TIC en España. Los datos para el estudio son estimativos dentro de la realidad, debido a que la intención no es saber estos valores reales sino el estudio teórico-práctico del método y su aplicación para el calculo del ROI. El estudio estadístico del la red social se realizo durante tres meses y se obtuvo el progreso de uso de la red social en eventos tales como: número de participantes activos, mensajes publicados, archivos subidos, grupos activos y tipos y plataformas de acceso. Del estudio de los datos estadísticos de estos eventos se obtuvieron indicadores de participación, actividad y conocimiento de la red social que son útiles par el calculo del ROI. En conclusión, se demuestran las mejoras que ofrece el social media en campos como la comunicación interna, gestión del conocimiento, formación e innovación. Y gracias a estas mejoras el aumento de la productividad y eficiencia del profesional y asimismo un potencial retorno de la inversión (ROI). ABSTRACT. This paper deals with the study of the implementation and development of the different platforms of social media: social networks and knowledge, blogs and collaborative tools in the corporate enterprise level. The study collects information technology consulting, articles and several social media platforms and an investigation into the question raised is performed. Analysis of 42 surveys of professionals from two big companies telecommunications in Spain are included. These two companies have one about 28000 employees and another with more than 300 employees at its Spanish subsidiaries. Both have a strong international presence. These two companies differ from other companies in the telecommunications sector they are betting in the implementation of social media in their internal processes. Furthermore, the study and analysis of usage statistics and a series of surveys on the wall of the corporate social network of a multinational telecommunications is included for three months. A new social culture enterprise is presented innovative in areas such as knowledge management, internal communications, training and innovation. And a quantitative vision into implementation of social media in the processes of a company is offered. They develops an exhibition where shown the process of studying the different social media platforms and application areas in the company, the study of the legal aspects of your application and use and implementation and development. A theoretical and practical analysis also exposed of calculation of return on investment (ROI) and finally an analysis of the information collected in surveys and statistical study of corporate social network. The survey data were analyzed using descriptive statistics based on graphs and contingency tables where waste and total percentages are calculated for analyze the dependence between the social media, efficiency, productivity and income statement, plus analysis of the contribution of social media on the mission, internal communication and knowledge management in the company. Also Calculations of chi-square distributions are conducted to demonstrate the dependence between of productivity and social media and the GAP that relates the importance and satisfaction level in social media. The theoretical and practical analysis parameters are the benefits, costs, flexibility and risk. The benefits are linked to productivity, knowledge management, human capital and internal processes. The costs are linked the software licensing, management, implementation and training. Based on these parameters was performed the study of a business model that represents a large ICT company in Spain. The data for the study are estimates within the reality, because the intention is not to know these real values but the theoretical and practical study and application of the method for calculating the ROI. Statistical analysis of the social network was made during or three months and was obtained the progress of social network use at events such as: number of active participants, messages posted, files uploaded, active groups and types and access platforms. Into study of statistical data of these events were obtained indicators of participation, activity and knowledge of the social network that are useful for calculating the ROI. In conclusion, the improvements offered by the social media in areas such as internal communication, knowledge management, training and innovation are shown. And thanks to these improvements increase the productivity and efficiency of professional and also a potential return on investment (ROI).

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Cell adhesion and the integrin-linked kinase regulate the LEF-1 and β-catenin signaling pathways

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the β1 and β3 integrin subunits and whose kinase activity is modulated by cell–extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell–cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of β-catenin to the nucleus, formation of a complex between β-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/β-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1/β-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/β-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell–matrix interactions and cell–cell adhesion as well as components of the Wnt signaling pathway.

Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize α2–6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of α2–6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous α2–6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein–Barr virus-transformed lymphoblasts which express high levels of α2–6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked during in vitro activation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked during in vivo activation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.

Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.

Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This ω-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein “pocket” binding activity, and do not suppress colony growth of RB(−) cells. In contrast, two low-penetrant alleles (661W and “deletion of codon 480”) retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, “deletion of RB exon 4,” showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(−) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

Thioredoxin-linked mitigation of allergic responses to wheat

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S—S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the α and β fractions were most potent, followed by the γ and ω types. Thioredoxin mitigated the allergenicity associated with the major protein fractions—i.e, the gliadins (including the α, β, and γ types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP–thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Identification of a structural determinant for resistance to β-lactam antibiotics in Gram-positive bacteria

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to β-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for β-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple β-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the “sensitive” form of PBP2x produces mutants similar, in terms of β-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for β-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of β-lactam antibiotics is lost.

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum of Saccharomyces cerevisiae: In Vivo and In Vitro Evidence

It has been proposed that synthesis of β-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparent Km of 46 μM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.