Ectopically expressed olfactory receptors OR51E1 and OR51E2 suppress proliferation and promote cell death in a prostate cancer cell line.

Olfactory receptors (ORs), the largest family of G protein-coupled receptors, are expressed in the nasal epithelium where they mediate the sense of smell. However, ORs are also found in other non-nasal tissues, but the role of these ectopic ORs in cell signaling, proliferation, and survival is not well understood. Here, using an inducible expression system in the lymph node carcinoma of the prostate (LNCaP) cell line, we investigated two ectopic ORs, OR51E1 and OR51E2, which have been shown to be upregulated in prostate cancer. We found that, consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short-chain to medium-chain organic acids (C3-C9) but not by acetate. OR51E2 responded to acetate and propionate but not to the longer chain organic acids. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Most significantly, overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of another ectopic OR OR2AT4, ß2-adrenergic receptor, or treatment of cells with forskolin did not suppress cell proliferation, indicating that a rise in cAMP is not sufficient to induce cytostasis. Overexpression of OR51E1 caused an upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining, and stimulated extracellular signal-regulated protein kinases 1 and 2. Overexpression and/or activation of OR51E1 did not affect human embryonic kidney 293 cell proliferation, indicating that cytotoxicity of OR51E1/OR51E2 is specific for LNCaP cells. Together, our results further our understanding of prostate cancer etiology and suggest that ectopic ORs may be useful therapeutic targets.

A Gs-RhoGEF interaction: An old G protein finds a new job.

The heterotrimeric G proteins are known to have a variety of downstream effectors, but Gs was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated Gs can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by Gs-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gαs subfamily regulates activity of Rho GTPases extends our understanding of Gαs activity and establishes RhoGEF coupling as a universal Gα function.

The regulatory G protein signaling complex, Gß5-R7, promotes glucose- and extracellular signal-stimulated insulin secretion.

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gß5-R7, a protein complex consisting of the atypical G protein ß subunit Gß5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit ß 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gß5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gß5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Emerging roles of the single EF-hand Ca2+ sensor tescalcin in the regulation of gene expression, cell growth and differentiation.

Tescalcin (TESC, also known as calcineurin-homologous protein 3, CHP3) is a 24-kDa EF-hand Ca2+-binding protein that has recently emerged as a regulator of cell differentiation and growth. The TESC gene has also been linked to human brain abnormalities, and high expression of tescalcin has been found in several cancers. The expression level of tescalcin changes dramatically during development and upon signal-induced cell differentiation. Recent studies have shown that tescalcin is not only subjected to up- or down-regulation, but also has an active role in pathways that drive cell growth and differentiation programs. At the molecular level, there is compelling experimental evidence showing that tescalcin can directly interact with and regulate the activities of the Na+/H+ exchanger NHE1, subunit 4 of the COP9 signalosome (CSN4) and protein kinase glycogen-synthase kinase 3 (GSK3). In hematopoetic precursor cells, tescalcin has been shown to couple activation of the extracellular signal-regulated kinase (ERK) cascade to the expression of transcription factors that control cell differentiation. The purpose of this Commentary is to summarize recent efforts that have served to characterize the biochemical, genetic and physiological attributes of tescalcin, and its unique role in the regulation of various cellular functions.

Regulator of G-protein signaling Gß5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.

In pancreatic ß cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in ß cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gß5, such that the knockout of the Gnb5 gene results in degradation of all R7 subunits. We found that Gnb5 knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gß5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in Gnb5-/- MIN6 cells showed that cAMP, diacylglycerol, or Ca2+ levels were not significantly affected. There was no reduction in the amplitude of free Ca2+ responses in islets from the Gnb5-/- mice, but the frequency of Ca2+ oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of Gnb5 impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in Gnb5-/- cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gß5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic ß-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gß5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.

Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor.

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth and is classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with muscarinic M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than muscarinic agonists oxotremorin-M (Oxo-M) or carbachol (CCh), although all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo-M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in human embryonic kidney cell line 293T (HEK293T) or mouse insulinoma (MIN6) cells. Pilocarpine also fails to stimulate insulin secretion and, instead, antagonizes the insulinotropic effect of Oxo-M and CCh-induced Ca2+ upregulation; however, in HEK293T or Chinese hamster ovary-K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another cognate G protein-coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R but not M3R. Moreover, pilocarpine blocked CCh-stimulated PIP2 hydrolysis in M3R-overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates extracellular regulated kinase 1/2 in MIN6 cells. The stimulatory effect on extracellular regulated kinase (ERK1/2) was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward ß-arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level, and signaling pathway downstream of this receptor.

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gß subunit, Gß5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gß5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gß5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.

Regulation of Cop9 signalosome activity by the EF-hand Ca2+-binding protein tescalcin.

The Ca(2+)-binding protein tescalcin is known to be involved in hematopoietic cell differentiation; however, this mechanism is poorly understood. Here, we identify CSN4 (subunit 4 of the COP9 signalosome) as a novel binding partner of tescalcin. The COP9 signalosome (CSN) is a multiprotein complex that is essential for development in all eukaryotes. This interaction is selective, Ca(2+)-dependent and involves the PCI domain of CSN4 subunit. We then investigated tescalcin and CSN activity in human erythroleukemia HEL and promyelocytic leukemia K562 cells and find that phorbol 12-myristate 13-acetate (PMA)-induced differentiation, resulting in the upregulation of tescalcin, coincides with reduced deneddylation of cullin-1 (Cul1) and stabilization of p27(Kip1) - molecular events that are associated with CSN activity. The knockdown of tescalcin led to an increase in Cul1 deneddylation, expression of F-box protein Skp2 and the transcription factor c-Jun, whereas the levels of cell cycle regulators p27(Kip1) and p53 decreased. These effects are consistent with the hypothesis that tescalcin might play a role as a negative regulator of CSN activity towards Cul1 in the process of induced cell differentiation.

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gß5-RGS7 complex.

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gß5-RGS7, the permanently associated heterodimer of G protein ß-subunit Gß5 and RGS7, a regulator of G protein signaling. Gß5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gß5-RGS7. In addition to the i3 loop, interaction of M3R with Gß5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gß5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gß5-RGS7 requires helix 8 and the central portion of the i3 loop.

Differential effects of the Gß5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ influx and release.

The G protein ß subunit Gß5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gß5-RGS7 complex attenuates Ca(2+) signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca(2+) entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca(2+) release, Gß5-RGS7 enhanced Ca(2+) influx. This novel effect of Gß5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gß5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gß5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca(2+) channel. We also compared the action of Gß5-RGS7 on M3R-induced Ca(2+) influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gß5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gß5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca(2+) response was enhanced by Gß5-RGS7, attributed to, in part, by the relatively small Ca(2+) release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gß5-RGS7 complex.

Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy).

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

Multiple opsins in a reef-building coral, Acropora millepora.

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1-6) from a coral species Acropora millepora, including three novel opsins (acropsins 4-6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca2+signaling light-dependently.

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

Targeted deletion of one or two copies of the G protein ß subunit Gß5 gene has distinct effects on body weight and behavior in mice.

We investigated the physiological role of Gß5, a unique G protein ß subunit that dimerizes with regulators of G protein signaling (RGS) proteins of the R7 family instead of Gγ. Gß5 is essential for stability of these complexes, so that its knockout (KO)causes degradation of the entire Gß5-R7 family. We report that the Gß5-KO mice remain leaner than the wild type (WT) throughout their lifetime and are resistant to a high-fat diet. They have a 5-fold increase in locomotor activity, increased thermogenesis, and lower serum insulin, all of which correlate with a higher level of secreted epinephrine. Heterozygous (HET) mice are 2-fold more active than WT mice. Surprisingly, with respect to body weight, the HET mice display a phenotype opposite to that of the KO mice: by the age of 6 mo, they are ≥ 15% heavier than the WT and have increased adiposity, insulin resistance, and liver steatosis. These changes occur in HET mice fed a normal diet and without apparent hyperphagia, mimicking basic characteristics of human metabolic syndrome. We conclude that even a partial reduction in Gß5-R7 level can perturb normal animal metabolism and behavior. Our data on Gß5 haploinsufficient mice may explain earlier observations of genetic linkage between R7 family mutations and obesity in humans.

Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage.

Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

Molecular organization of the complex between the muscarinic M3 receptor and the regulator of G protein signaling, Gbeta(5)-RGS7.

The complex of the regulator of G protein signaling (RGS), Gbeta(5)-RGS7, can inhibit signal transduction via the M3 muscarinic acetylcholine receptor (M3R). RGS7 consists of three distinct structural entities: the DEP domain and its extension DHEX, the Ggamma-like (GGL) domain, which is permanently bound to Gbeta subunit Gbeta(5), and the RGS domain responsible for the interaction with Galpha subunits. Inhibition of the M3R by Gbeta(5)-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the third intracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction between the Gbeta(5) and DEP domains, which suggested that the Gbeta(5)-RGS7 dimer could alternate between the "open" and "closed" conformations. Here, we identified point mutations that weaken DEP-Gbeta(5) binding, presumably stabilizing the open state, and tested their effects on the interaction of Gbeta(5)-RGS7 with the M3R. We found that these mutations facilitated binding of Gbeta(5)-RGS7 to the recombinant third intracellular loop of the M3R but did not enhance its ability to inhibit M3R-mediated Ca(2+) mobilization. This led us to the idea that the M3R can effectively induce the Gbeta(5)-RGS7 dimer to open; such a mechanism would require a region of the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts with Gbeta(5)-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-type Gbeta(5)-RGS7; however, the open mutant of Gbeta(5)-RGS7 was able to inhibit signaling by the truncated M3R. The GST fusion of the M3R C-tail could not bind to wild-type Gbeta(5)-RGS7 but could associate with its open mutant as well as with the separated recombinant DEP domain or Gbeta(5). Taken together, our data are consistent with the following model: interaction of the M3R with Gbeta(5)-RGS7 causes the DEP domain and Gbeta(5) to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop, thereby inhibiting M3R-mediated signaling.

Tescalcin is an essential factor in megakaryocytic differentiation associated with Ets family gene expression.

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.

Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions.

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.