Four novel splice variants of sulfonylurea receptor 1

ATP-sensitiveK⁺ (K_ATP) channels are composed of pore-formingKir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pigSUR1. Three splice forms originate from alternative splicing of theregion coding for NBF1 and lack exons 17 (SUR117), 19 (SUR119),or both (SUR11719). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31-39 containing the last twotransmembrane segments and the COOH terminus of SUR1. RT-PCR analysisshowed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy usingenhanced green fluorescent protein-tagged SUR or Kir6.x did not provideany evidence for involvement of these splice forms in themitochondrial K_ATP channel. Only SUR1 and SUR117 showed high-affinity binding of glibenclamide (K_d 2 nM in the presence of 1 mM ATP) and formed functional K_ATPchannels upon coexpression with Kir6.2.

     

Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility

Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10(-12) to 10(-5)) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression.     

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.     

Blocking GRP/GRP-R signaling decreases expression of androgen receptor splice variants and inhibits tumor growth in castration-resistant prostate cancer

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone – Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth.     

Differential trafficking of GluR7 kainate receptor subunit splice variants

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.     

Phosphorylation and nuclear processing of the androgen receptor.

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.     

Expression of OB receptor splice variants during prenatal development of the mouse

In adult animals, signaling through the leptin receptor (OB-R) has been shown to play a critical role in fat metabolism. However, it is not known when these receptors are first expressed and what their role may be during embryonic development. To date, at least 6 splice variants of the OB-R have been identified. Although the function of each of these individual splice variants are unknown, only one of them, ob-rL ,encodes a receptor with a long intracellular domain that is implicated in OB-R signaling. In this study we have used in situ hybridization to examine the localization of OB-R splice variants during embryonic development of C57B1/6J mice. Using a probe, ob-r, that recognizes all of the splice variants, ob-r mRNA was found to be distributed in developing bone, mesenchyme, notochord and liver. In addition, epithelial structures including leptomeninges, choroid plexi and hair follicles also expressed ob-r. No ob-r mRNA was detected in the CNS. ob-rL, expression was only detected in notochord, bone and mesenchyme. The differential expression of these two mRNA isoforms suggests that the extracellular and intracellular domains of the OB receptor perform different biological functions.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Opposite effects of alternative TZF spliced variants on androgen receptor

We previously demonstrated that testicular zinc-finger protein (TZF) was a corepressor of the androgen receptor (AR). In the present study, we further showed that TZF-L, an alternative spliced variant of TZF, enhanced transactivation function of AR. Deletion analysis of TZF-L revealed that its N-terminus, which almost corresponded to that of TZF, but not its C-terminus was able to interact with AR. Additional analysis suggested that TZF and TZF-L were able to form both homodimers and heterodimers. TZF-L inhibited the homodimer formation of TZF and the intranuclear dot formation of TZF. We propose that in the unique regulation system of AR-mediated transactivation, two spliced isoforms of TZF act as coactivator and corepressor, respectively.