Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Identifying novel protein complexes in cancer cells using epitope-tagging of endogenous human genes and affinity-purification mass spectrometry.

Affinity-purification mass spectrometry (AP-MS) is the preeminent technique for identification of eukaryotic protein complexes in vivo. AP-MS workflows typically express epitope-tagged bait proteins, immunopurify, and then identify associated protein complexes using mass spectrometry. However, challenges of existing strategies include the construction of expression vectors for large open reading frames and the possibility that overexpression of bait proteins may result in expression of nonphysiological levels of the bait protein with concomitant perturbation of endogenous protein complexes. To address these issues, we use human cell lines with epitope-tagged endogenous genes as AP-MS substrates to develop a platform that we call "knock-in AP-MS", thereby avoiding the challenges of expression vector construction and ensuring that expression of tagged proteins is driven by endogenous regulatory mechanisms. Using three different bait genes (MRE11A, DNMT1 and APC), we show that cell lines expressing epitope-tagged endogenous genes make good substrates for sensitive and reproducible identification of protein interactions using AP-MS. In particular, we identify novel interactors of the important oncoprotein Adenomatous Polyposis Coli (APC), including an interaction with Flightless-1 homologue (FLII) that is enriched in nuclear fractions.

Daphnia HR96 is a promiscuous xenobiotic and endobiotic nuclear receptor.

Daphnia pulex is the first crustacean to have its genome sequenced. The genome project provides new insight and data into how an aquatic crustacean may respond to environmental stressors, including toxicants. We cloned Daphnia pulex HR96 (DappuHR96), a nuclear receptor orthologous to the CAR/PXR/VDR group of nuclear receptors. In Drosophila melanogaster, (hormone receptor 96) HR96 responds to phenobarbital exposure and has been hypothesized as a toxicant receptor. Therefore, we set up a transactivation assay to test whether DappuHR96 is a promiscuous receptor activated by xenobiotics and endobiotics similar to the constitutive androstane receptor (CAR) and the pregnane X-receptor (PXR). Transactivation assays performed with a GAL4-HR96 chimera demonstrate that HR96 is a promiscuous toxicant receptor activated by a diverse set of chemicals such as pesticides, hormones, and fatty acids. Several environmental toxicants activate HR96 including estradiol, pyriproxyfen, chlorpyrifos, atrazine, and methane arsonate. We also observed repression of HR96 activity by chemicals such as triclosan, androstanol, and fluoxetine. Nearly 50% of the chemicals tested activated or inhibited HR96. Interestingly, unsaturated fatty acids were common activators or inhibitors of HR96 activity, indicating a link between diet and toxicant response. The omega-6 and omega-9 unsaturated fatty acids linoleic and oleic acid activated HR96, but the omega-3 unsaturated fatty acids alpha-linolenic acid and docosahexaenoic acid inhibited HR96, suggesting that these two distinct sets of lipids perform opposing roles in Daphnia physiology. This also provides a putative mechanism by which the ratio of dietary unsaturated fats may affect the ability of an organism to respond to a toxic insult. In summary, HR96 is a promiscuous nuclear receptor activated by numerous endo- and xenobiotics.

Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

Coiled-coil tag-probe labeling methods for live-cell imaging of membrane receptors.

Tag-probe labeling methods have advantages over conventional fusion with fluorescent proteins in terms of smaller labels, surface specificity, availability of pulse labeling, and ease of multicolor labeling. With this method, the gene of the target protein is fused with a short tag sequence, expressed in cells, and the protein is labeled with exogenous fluorescent probes that specifically bind to the tag. Various labeling principles, such as protein-ligand interaction, peptide-peptide interaction, peptide-metal interaction, and enzymatic reactions, have been applied to the tag-probe labeling of membrane receptors. We describe our coiled-coil tag-probe method in detail, including the design and synthesis of the tag and probe, labeling procedures, and observations by confocal microscopy. Applications to the analysis of receptor internalization and oligomerization are also introduced.

Quantitative measurement of phosphatidylinositol 3,4,5-trisphosphate.

The activation of class I phosphoinositide 3-kinases (PI(3)Ks) by cell surface receptors represents the initiation of a large and complex signaling network that couples many growth factors, antigens, and inflammatory stimuli to important cellular responses, such as cell growth, survival, and movement. The most direct measurement of class I PI(3)K activity in cells is the rate of production of its lipid product, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. This chapter describes in detail two approaches used to estimate the levels of PtdIns(3,4,5)P(3) in cells. One approach uses radiotracer labeling of cells, lipid extraction, deacylation, and subsequent quantitation of phosphoinositides by anion-exchange high-performance liquid chromatography. The second approach uses a novel, nonradioactive assay in which the cellular lipids are extracted, phosphoinositides are enriched through binding to a neomycin matrix, dried onto a nitrocellulose membrane, and PtdIns(3,4,5)P(3) quantified by a protein-lipid overlay approach using a GRP(1) PH domain probe.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface.

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.

Drosophila nuclear receptor E75 is a thiolate hemoprotein.

Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.

Proteomic analysis of steady-state nuclear hormone receptor coactivator complexes.

We report our initial efforts in the analysis of endogenous nuclear receptor coactivator complexes as a research bridging strand of the Nuclear Receptor Signaling Atlas (NURSA) (www.NURSA.org). A proteomic approach is used to systematically isolate a variety of coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry. We have isolated and identified seven coactivator complexes including the p160 steroid receptor coactivator family, cAMP response element binding protein-binding protein, p300, coactivator of activating protein-1 and estrogen receptors, and E6 papillomavirus-associated protein. The newly identified coactivator-associated proteins provide unbiased clues and links for understanding of the endogenous hormone receptor coregulator network and its regulation. We hope that the electronic availability of these data to the general scientific community will facilitate generation and testing of new hypotheses to further our understanding of nuclear receptor signaling and coactivator functions.

Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain.

NOR-1 is an orphan member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, fatty acids, retinoic acids, and other lipophilic molecules. The NOR-1 subfamily (NR4), composed also of Nurr1 and Nurr77, has been implicated in cell proliferation, differentiation, apoptosis, chondrosarcomas, inflammation, and atherogenesis. The NOR-1 receptor is an orphan ligand receptor which acts over gene transactivation. No ligands, if such in fact exist, are known for this receptor. Recently, the three-dimensional structure of the homolog receptor Nurr1 has been solved using protein crystallography techniques. Surprisingly, the structure does not present either a typical cavity for ligand binding or a classical co-factor binding site in the ligand binding domain (LBD). To allow for structural studies of other members of NR4 subfamily, we have subcloned, overexpressed in Escherichia coli cells, purified, and characterized the rat orphan nuclear receptor NOR-1 LBD domain. We obtained NOR-1 LBD at a high degree of purity and with an overall yield of 3 mg/L of culture media. CD spectroscopic analysis shows a high alpha-helical secondary structure content (52%), similar to that of Nurr 1 LBD three-dimensional structure. Thermal denaturation monitored by UV absorption and CD spectroscopy suggests proper folding of recombinant NOR-1 LBD.

Bitter gourd (Momordica charantia) extract activates peroxisome proliferator-activated receptors and upregulates the expression of the acyl CoA oxidase gene in H4IIEC3 hepatoma cells.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-dependent transcription factor that regulates the expression of genes involved in lipid metabolism and transport. Ligands/activators of PPARalpha, like fibrate-type drugs, may have hypolipidemic effects. To identify food that contains activators of PPARalpha, a transactivation assay employing a clone of CHO-K1 cells stably transfected with a (UAS)(4)-tk-alkaline phosphatase reporter and a chimeric receptor of Gal4-rPPARalpha LBD was used to screen ethyl acetate (EA) extracts of a large variety of food materials. It was found that the EA extract of bitter gourd (Momordica charantia), a common oriental vegetable, activated PPARalpha to an extent that was equivalent to or even higher than 10 microM Wy-14643, a known ligand of PPARalpha. This extract also activated PPARgamma to a significant extent which was comparable to 0.5 microM BRL-49653. The activity toward PPARalpha was mainly in the soluble fraction of the organic solvent. The EA extract prepared from the whole fruit showed significantly higher activity than that from seeds or flesh alone. The bitter gourd EA extract was then incorporated into the medium for treatment of a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, for 72 h. Treated cells showed significantly higher activity of acyl CoA oxidase and higher expressions of mRNA of this enzyme and fatty acid-binding protein, indicating that the bitter gourd EA extract was able to act on a natural PPARalpha signaling pathway in this cell line. It is thus worth further investigating the PPAR-associated health benefits of bitter gourd.

Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay.

The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7alpha-hydroxylase, which in turn regulates bile acid production and cholesterol excretion. To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library. Three human FXR cDNA, designated FXR20, FXR33, and FXR53 cDNA, were subcloned and ligated into a pET28a expression vector. Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography. Approximately 5 mg of FXR33 (1-182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR53 (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity. The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N1-labeled anti-His antibody. From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR33 and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 microM). The ligand-binding domain of FXR (FXR33) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR53) or the DNA-binding domain of FXR (FXR20).

Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism.

The polypeptide hormone stanniocalcin (STC) is widely expressed in mammalian tissues. STC acts locally in kidney and gut to modulate calcium and phosphate excretion, and its overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate. STC has also been linked to cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress. In this report we have characterized the STC receptor and the functional targeting of ligand and receptor to mitochondria. For receptor binding analysis, a stanniocalcin-alkaline phosphatase fusion protein was engineered. Subsequent binding assays using the fusion protein indicated that kidney and liver contained the highest number of binding sites with affinities of 0.8 and 0.25 nm, respectively. Intriguingly, purified mitochondria from both tissues yielded similar high affinity binding sites. Fractionation analysis revealed that the majority of binding sites were localized to the inner mitochondrial membrane. In further studies, we characterized the time course of STC-alkaline phosphatase fusion protein sequestration by intact mitochondria. In situ ligand binding also revealed discrete, displaceable binding to plasma membranes and mitochondria of nephron cells and liver hepatocytes. The existence of mitochondrial receptors prompted a similar search for the ligand. Immunogold electron microscopy revealed that STC was preferentially concentrated in the mitochondria of all nephron segments targeted by STC. Subcellular fractionation revealed that >90% of cellular STC immunoreactivity was mitochondrial, confined to the inner matrix, and similar in size to recombinant STC (50 kDa). In functional studies, recombinant STC had concentration-dependent stimulatory effects on electron transfer by sub-mitochondrial particles. Collectively the evidence implies a role for STC in cell metabolism.

Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane.

We cloned a approximately 70 kDa rat mitochondrial outer membrane protein (OM70) with a sequence identity of 28.1% and 20.1% with N. crassa and S. cerevisiae Tom70, respectively. Even with this low sequence identity, however, the proteins share a remarkable structural similarity: they have 7-10 tetratricopeptide repeat motifs and are anchored to the outer membrane through the N-terminal transmembrane domain with the bulk portion located in the cytosol. Antibodies against OM70 inhibited import of preproteins, such as the ADP/ATP carrier and rTOM40, that use internal targeting signals but not the import of cleavable presequence-containing preproteins. Blue native gel electrophoresis and immunoprecipitation of digitoninsolubilized mitochondrial outer membranes revealed that OM70 was loosely associated with the approximately 400 kDa translocase complex of the mitochondrial outer membrane, which contains rTOM22 and rTOM40. A yeast two-hybrid system demonstrated that OM70 interacted with rTOM20 and rTOM22 through the cytoplasmic domains. Thus, OM70 is a functional homologue of fungal Tom70 and functions as a receptor of the preprotein import machinery of the rat mitochondrial outer membrane. Furthermore, the N-terminal 66 residue region of OM70, which comprises a hydrophilic 41 residue N-terminal domain, a 22 residue transmembrane domain and three arginine residues, is sufficient to act as a mitochondria-targeting signal, and the arginine cluster is crucial for this function.

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48.

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.

Cyclooxygenase-two (COX-2) modulates proliferation in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis is a locally invasive soft tissue lesion. Seventy-five per cent of cases harbor a somatic mutation in either the APC or beta-catenin genes, resulting in beta-catenin protein stabilization. Cyclooxygenase-2 (COX-2) is an enzyme involved in prostaglandin synthesis that modulates the formation of colonic neoplasia, especially in cases due to mutations resulting in beta-catenin stabilization. Human aggressive fibromatoses and lesions from the Apc+/Apc1638N mouse (a murine model for Apc-driven fibromatosis) demonstrated elevated COX-2 levels. COX-2 blockade either by the selective agent DFU or by non-selective COX blocking agents results in reduced proliferation in human tumor cell cultures. Breeding mice with Cox-2-/- mice resulted in no difference in number of aggressive fibromatoses formed, but in a smaller tumor size, while there was a decrease in number of GI lesions by 50%. Mice fed various COX blocking agents also showed a decline in tumor size. COX-2 expression was regulated by tcf-dependent transcription in this lesion. COX-2 partially regulates proliferation due to beta-catenin stabilization in aggressive fibromatosis. Although COX blockade alone does not cause tumor regression, this data suggests that it may have a role as an adjuvant therapy to slow tumor growth in this lesion.

Retinoic acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new class of nuclear receptor isoforms.

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

A human importin-beta family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins.

Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-beta family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins.

Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications.

During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain.

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an alpha-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 10(5) times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase-nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.