Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development.

Designed Modular Proteins as Scaffolds To Stabilize Fluorescent Nanoclusters.

Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.

Seipin deficiency increases chromocenter fragmentation and disrupts acrosome formation leading to male infertility.

The Berardinelli-Seip congenital lipodystrophy type 2 (Bscl2, seipin) gene is involved in adipogenesis. Bscl2-/- males were infertile but had normal mating behavior. Both Bscl2-/- cauda epididymis sperm count and sperm motility were ~20×less than control. Bscl2-/- seminiferous tubules had relatively normal presence of spermatogonia and spermatocytes but had reduced spermatids and sperm. Spatiotemporal expression analyses in Bscl2+/+ testes demonstrated prominent Bscl2 transcriptional activity in spermatocytes with a plateau reached around postnatal day 28. Seipin protein localization was most abundant in postmeiotic spermatids, suggesting translational repression of Bscl2 mRNA in spermatocytes. In situ end-labeling plus detected increased spermatid apoptosis in Bscl2-/- testis and annexin V detected increased percentage of positive Bscl2-/- round spermatids compared with control. Immunofluorescence of marker proteins synaptonemal complex proteins 3 and 1 (SYCP3 and SYCP1), and H3K9me3 (histone H3 trimethylated at lysine 9) in germ cell spreads detected normal meiotic chromosome pairing and homologous chromosome synapsis in Bscl2-/- spermatocytes, but significantly increased percentages of round spermatids with chromocenter fragmentation and late spermatids and sperm with chromatin vacuoles, indicating defective chromatin condensation in Bscl2-/- spermatids. Bscl2-/- late spermatids were disorganized within the seminiferous epithelium, despite normal appearance of Sertoli cells detected by vimentin immunofluorescence. Peanut agglutinin staining revealed various abnormalities of acrosomes in Bscl2-/- late spermatids, including the absence, irregular-shaped, and fragmented acrosomes, indicating defective acrosome formation in Bscl2-/- late spermatids, which may affect late spermatid orientation in the seminiferous epithelium. Mitotracker strongly stained the midpiece of control sperm but only very weakly labeled the midpiece of Bscl2-/- sperm, indicating defective mitochondrial activity that most likely contributed to reduced Bscl2-/- sperm motility. These data demonstrate novel roles of seipin in spermatid chromatin integrity, acrosome formation, and mitochondrial activity. Increased spermatid apoptosis, increased chromocenter fragmentation, defective chromatin condensation, abnormal acrosome formation, and defective mitochondrial activity contributed to decreased sperm production and defective sperm that resulted in Bscl2-/- male infertility.

Splice isoforms of phosducin-like protein control the expression of heterotrimeric G proteins.

Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein ß subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gß and the assembly of Gß and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gß. As a result, the cellular levels of Gß and Gγ, which depends on Gß for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins.

Suppression of adipogenesis by pathogenic seipin mutant is associated with inflammatory response.

BACKGROUND: While pathogenic mutations in BSCL2/Seipin cause congenital generalized lipodystrophy, the underlying mechanism is largely unknown. In this study, we investigated whether and how the pathogenic missense A212P mutation of Seipin (Seipin-A212P) inhibits adipogenesis. METHODOLOGY/RESULTS: We analyzed gene expression and lipid accumulation in stable 3T3-L1 cell lines expressing wild type (3T3-WT), non-lipodystrophic mutants N88S (3T3-N88S) and S90L (3T3-S90L), or lipodystrophic mutant A212P Seipin (3T3-A212P). When treated with adipogenic cocktail, 3T3-WT, 3T3-N88S and 3T3-S90L cells exhibited proper differentiation into mature adipocytes, indistinguishable from control 3T3-L1 cells. In contrast, adipogenesis was significantly impaired in 3T3-A212P cells. The defective adipogenesis in 3T3-A212P cells could be partially rescued by either PPARγ agonist or PPARγ overexpression. Gene expression profiling by microarray revealed that inhibition of adipogenesis was associated with activation of inflammatory genes including IL-6 and iNOS. We further demonstrated that Seipin-A212P expression at pre-differentiation stages significantly activated inflammatory responses by using an inducible expression system. The inflammation-associated inhibition of adipogenesis could be rescued by treatment with anti-inflammatory agents. CONCLUSIONS: These results suggest that pathogenic Seipin-A212P inhibits adipogenesis and the inhibition is associated with activation of inflammatory pathways at pre-differentiation stages. Use of anti-inflammatory drugs may be a potential strategy for the treatment of lipodystrophy.

Identification and partial characterization of Rhizopus nigricans Gß proteins and their expression in the presence of progesterone.

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gß subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gß subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gß from other filamentous fungi, but not from yeast. The presence of more than one Gß subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gß subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gß subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.

mGluR6 deletion renders the TRPM1 channel in retina inactive.

In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G(o), which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates Gα(o), Gß(3), and Gγ(13) showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor.

Human G-protein gamma 7 in extrahepatic cholangiocarcinoma and its clinicopathological significance.

BACKGROUND AND OBJECTIVES: Several studies have found a down-regulated G-gamma 7 gene in gastrointestinal tract cancers. We evaluated the expression and clinicopathological significance of the human G protein gamma 7 (G-gamma 7) in human extra-hepatic cholangiocarcinoma (EHCC). METHODS: The expression of G-gamma 7 expression was studied in 21 patients with EHCC. G-gamma 7 mRNA expression was tested by using RealTime reverse transcription polymerase chain reaction (RT-PCR).To visualize the localization of G-gamma 7, an immunohistochemistry study was also performed. The G-gamma 7 expression was compared among cancer tissues, peri-cancerous bile duct tissues and normal bile duct tissues. The clinicopathological significance of G-gamma 7 expression was also studied. RESULTS: Expression of G-gamma 7 mRNA and protein were significantly lower in EHCC tissue than in pericancerous bile duct tissue and normal bile duct tissues. G-gamma 7 mRNA and protein expression were significantly lower in poorly differentiated EHCC tissues than in moderate differentiated and well differentiated EHCC tissues (P<.01). There was no significant correlation between G-gamma 7 expression and host factors such as age, gender, clinical staging or the status of preoperative hepatic function CONCLUSIONS: EHCC has a down-regulated expression of G-gamma 7. Reduced expression of G-gamma 7 is associated with the histological grade of EHCC and may prove to be a useful marker for predicting the prognosis of human EHCC.

The pattern of expression of guanine nucleotide-binding protein beta3 in the retina is conserved across vertebrate species.

Guanine nucleotide-binding protein beta3 (GNB3) is an isoform of the beta subunit of the heterotrimeric G protein second messenger complex that is commonly associated with transmembrane receptors. The presence of GNB3 in photoreceptors, and possibly bipolar cells, has been confirmed in murine, bovine and primate retinas [Lee RH, Lieberman BS, Yamane HK, Bok D, Fung BK (1992) J Biol Chem 267:24776-24781; Peng YW, Robishaw JD, Levine MA, Yau KW (1992) Proc Natl Acad Sci U S A 89:10882-10886; Huang L, Max M, Margolskee RF, Su H, Masland RH, Euler T (2003) J Comp Neurol 455:1-10]. Studies have indicated that a mutation in the GNB3 gene causes progressive retinopathy and globe enlargement (RGE) in chickens. The goals of this study were to (1) examine the expression pattern of GNB3 in wild-type and RGE mutant chickens, (2) characterize the types of bipolar cells that express GNB3 and (3) examine whether the expression of GNB3 in the retina is conserved across vertebrate species. We find that chickens homozygous for the RGE allele completely lack GNB3 protein. We find that the pattern of expression of GNB3 in the retina is highly conserved across vertebrate species, including teleost fish (Carassius auratus), frogs (Xenopus laevis), chickens (Gallus domesticus), mice (Mus musculata), guinea-pigs (Cavia porcellus), dogs (Canis familiaris) and non-human primates (Macaca fasicularis). Regardless of the species, we find that GNB3 is expressed by Islet1-positive cone ON-bipolar cells and by cone photoreceptors. In some vertebrates, GNB3-immunoreactivity was observed in both rod and cone photoreceptors. A protein-protein alignment of GNB3 across different vertebrates, from fish to humans, indicates a high degree (>92%) of sequence conservation. Given that analogous types of retinal neurons express GNB3 in different species, we propose that the functions and the mechanisms that regulate the expression of GNB3 are highly conserved.

[The role of molecular chaperones in the assembly of heterotrimeric G proteins]. / Fonction des chaperonnes moléculaires dans l'assemblage des protéines G hétérotrimériques.

Extracellular signals received by G protein-coupled receptors (GPCRs) are transduced into intracellular responses following the activation of heterotrimeric G proteins. As their names suggests, they are composed of three subunits, Galpha and Gbetagamma, the latter being effectively treated as a single entity. The Gbetagamma dimer is assembled with the aid of a number of molecular chaperones in a tightly regulated process. The folding of nascent Gbeta1 is favoured by cellular chaperones such as PhLP-1 and CCT and the ER-resident protein DRiP78 plays an important role in the stability of nascent Ggamma2 subunits. However, much work remains to be done to completely understand the mechanisms underlying assembly of the heterotrimer.

Expression of Galpha14 in sweet-transducing taste cells of the posterior tongue.

BACKGROUND: "Type II"/Receptor cells express G protein-coupled receptors (GPCRs) for sweet, umami (T1Rs and mGluRs) or bitter (T2Rs), as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCbeta2-mediated release of stored Ca2+. Whereas Galphagus (gustducin) couples to the T2R (bitter) receptors, which Galpha-subunit couples to the sweet (T1R2 + T1R3) receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Galphaq family (q, 11, 14) in mouse taste buds. RESULTS: By RT-PCR, Galpha14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate), but not anterior (fungiform and palate) taste fields. Galphaq and Galpha11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Galpha14 mRNA is limited to Type II/Receptor cells in taste buds. Immunocytochemistry on vallate papillae using a broad Galphaq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCbeta2). This staining persists in Galphaq knockout mice and immunostaining with a Galpha11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Galpha14 is the dominant Galphaq family member detected. Immunoreactivity for Galpha14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Galpha14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. CONCLUSION: Galpha14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.

Prospects for retinal cone-targeted gene therapy.

Gene therapy strategies that target therapeutic genes to retinal cones are a worthy goal both because cone photoreceptor diseases are severely vision limiting and because many retinal diseases that do not affect cones directly eventually lead to cone loss, the reason for eventual blindness. Human achromatopsia is a genetic disease of cones that renders them nonfunctional but otherwise intact. Thus, animal models of achromatopsia were used in conjunction with adeno-associated virus (AAV) vectors whose serotype efficiently transduces cones and with a promoter that limits transgene expression to cones. In the Gnat2(cpfl3) mouse model of one genetic form of human achromatopsia, we were able to demonstrate recovery of normal cone function and visual acuity after a single subretinal treatment of vector that supplied wild-type Gnat2 protein to cones. This validates the overall strategy of targeting cones using recombinant viral vectors and justifies a more complete examination of animal models of cone disease as a prelude to considering a clinical gene therapy trial.

Microarray gene expression profiling of mouse brain mRNA in a model of lithium treatment.

OBJECTIVES: Even after five decades of use, the mood stabilizer lithium continues to be the mainstay of treatment for bipolar disorder in many countries. The mechanism of action for lithium, however, remains unclear. METHODS: In this study, microarray analysis was used to identify genes and cellular pathways that are altered in the mouse brain after treatment with lithium at human therapeutic concentrations. Mice received daily injections of lithium chloride for 7 consecutive days. Whole-brain total RNA was used as a template for microarray gene expression profiling. RESULTS: This study has identified 19 transcripts that are differentially expressed by four-fold when compared with control untreated mice. The altered expression of these genes was validated by quantitative PCR analysis with five genes showing significant differential expression. Lithium was found to significantly decrease the expression of metallothionein 3 (MT3), ATPase, Na/K transporting, alpha1 polypeptide (ATP1A1), transcription elongation factor B (SIII)-polypeptide 2 (TCEB2), proteasome subunit beta type 5 (PSMB5), and guanine nucleotide binding protein beta1 (GNB1). CONCLUSION: These genes are involved in a diverse range of biological functions, including maintaining metal ion homeostasis and chemical/electrical gradients across membranes, regulating RNA polymerase II, protein degradation, and G-protein-coupled signal transduction. These results indicate that lithium can regulate a large number of different cellular pathways in the brain. Understanding the molecular and cellular mechanisms by which lithium achieves its therapeutic action represents a valuable step in clarifying the pathophysiology of bipolar disorder.

Regional differences in taste bud distribution and alpha-gustducin expression patterns in the mouse fungiform papilla.

The regional differences between distribution patterns and alpha-gustducin expression patterns of the fungiform (FF) taste buds were investigated in the adult mouse, using hematoxylin-eosin staining and immunofluorescence histochemistry on the most anterior region of the tongue (the first millimeter) through the intermediate region of the tongue (the last 1-4 mm). Paraffin sections were prepared from the tip to posterior regions (anterior and intermediate region containing the FF taste buds) of the adult mouse tongue. Results indicate that there were significant regional differences in size and density of taste buds, the cell counts of the single taste bud, and the alpha-gustducin-immunoreactive taste buds between the 2 regions. The taste bud had a characteristic onion-like appearance, and the alpha-gustducin-immunoreactive cell was spindle shaped with elongated processes extending from the base to the pore of the taste buds. These results provide a detailed insight to better understand regional descriptions of mouse taste bud density and size and alpha-gustducin expression with the mouse model.

The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology.

Lipodystrophy is a disorder characterized by a loss of adipose tissue often accompanied by severe hypertriglyceridemia, insulin resistance, diabetes, and fatty liver. It can be inherited or acquired. The most severe inherited form is Berardinelli-Seip Congenital Lipodystrophy Type 2, associated with mutations in the BSCL2 gene. BSCL2 encodes seipin, the function of which has been entirely unknown. We now report the identification of yeast BSCL2/seipin through a screen to detect genes important for lipid droplet morphology. The absence of yeast seipin results in irregular lipid droplets often clustered alongside proliferated endoplasmic reticulum (ER); giant lipid droplets are also seen. Many small irregular lipid droplets are also apparent in fibroblasts from a BSCL2 patient. Human seipin can functionally replace yeast seipin, but a missense mutation in human seipin that causes lipodystrophy, or corresponding mutations in the yeast gene, render them unable to complement. Yeast seipin is localized in the ER, where it forms puncta. Almost all lipid droplets appear to be on the ER, and seipin is found at these junctions. Therefore, we hypothesize that seipin is important for droplet maintenance and perhaps assembly. In addition to detecting seipin, the screen identified 58 other genes whose deletions cause aberrant lipid droplets, including 2 genes encoding proteins known to activate lipin, a lipodystrophy locus in mice, and 16 other genes that are involved in endosomal-lysosomal trafficking. The genes identified in our screen should be of value in understanding the pathway of lipid droplet biogenesis and maintenance and the cause of some lipodystrophies.

Regulation of cardiac cAMP synthesis and contractility by nucleoside diphosphate kinase B/G protein beta gamma dimer complexes.

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

[Expression, loss of heterozygosity, and methylation of GNAT1 gene in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: In nasopharyngeal carcinoma (NPC), chromosome 3p21.3 is a high frequency deletion region. Evidences from both loss of heterozygosity (LOH) and functional studies showed that there may exists NPC-related tumor suppressor genes on 3p21.3. This study was to investigate the expression, LOH, and methylation of GNAT1 gene, which locates at 3p21.3, in NPC. METHODS: The expression of GNAT1 in 33 specimens of primary NPC and 15 specimens of chronic nasopharyngitis tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). LOH and promoter methylation status of GNAT1 were examined by microsatellites analysis and methylation-specific polymerase chain reaction (MSP). RESULTS: GNAT1 was expressed stably in all chronic nasopharyngitis tissues, while absent or down-regulated in 24 (72.7%) specimens of NPC (P=0.022). LOH analysis showed allele loss of GNAT1 in 3 (15%) specimens of NPC. LOH of GNAT1 was correlated to its expression level (P=0.016). Methylation analysis showed hypermethylation of GNAT1 promoter in all primary NPC tissues and in 12 (80%) specimens of chronic nasopharyngitis tissues. CONCLUSIONS: Expression of GNAT1 gene is down-regulated or absent in NPC tissues, which may relate to allele loss of GNAT1 in NPC, but not relate to its promoter hypermethylation. Hypermethylation of GNAT1 may not be oncogenic mechanisms of NPC.

Signalling complexes associated with adenylyl cyclase II are assembled during their biosynthesis.

We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta2-adrenergic receptors and heterotrimeric G proteins as part of a pre-assembled signalling complex. In this study, we further show that AC interacts with these proteins before it is targetted to the cell surface. Using a combination of approaches including bioluminescence resonance energy transfer (BRET) in concert with subcellular fractionation, we show that ACII and beta2AR initially interact in the ER. Further, dominant-negative Rab1 and Sar1 GTPases which block anterograde trafficking out of the ER have no effect on either ACII/receptor or ACII/Gbetagamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Galpha subunits in ACII signalling complexes suggesting it assembles into the complex at a slightly later stage. Thus, like Kir3.1 inwardly rectifying potassium channels, signalosomes containing ACII are formed during their biosynthesis and not in response to agonist at the cell surface.

G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation.

The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.

Disruption of type 5 adenylyl cyclase negates the developmental increase in Galphaolf expression in the striatum.

The two stimulatory G protein alpha subunits, Galphas and Galphaolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Galphas and Galphaolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Galphaolf, but not Galphas. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Galphaolf, but not Galphas.