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SUMMARY The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, β-catenin, can physically associate as a ternary complex. This association stabilizes β-catenin via HDAC6 deacetylase activity, which leads to activation of β-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased β-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein-protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4-6 weeks.

Distal renal tubular acidosis (dRTA), a kidney disease resulting in defective urinary acidification, can be caused by dominant or recessive mutations in the kidney Cl -/HCO 3 -anion exchanger (kAE1), a glycoprotein expressed in the basolateral membrane of a-intercalated cells. We compared the effect of two dominant (R589H and S613F) and two recessive (S773P and G701D) dRTA point mutations on kAE1 trafficking in Madin-Darby canine kidney (MDCK) epithelial cells. In contrast to wild-type (WT) kAE1 that was localized to the basolateral membrane, the dominant mutants (kAE1 R589H and S613F) were retained in the endoplasmic reticulum (ER) in MDCK cells, with a few cells showing in addition some apical localization. The recessive mutant kAE1 S773P, while misfolded and largely retained in the ER in non-polarized MDCK cells, was targeted to the basolateral membrane after polarization. The other recessive mutants, kAE1 G701D and designed G701E, G701R but not G701A or G701L mutants, were localized to the Golgi in both non-polarized and polarized cells. The results suggest that introduction of a polar mutation into a transmembrane segment resulted in Golgi retention of the recessive G701D mutant. When coexpressed, the dominant mutants retained kAE1 WT intracellularly, while the recessive mutants did not. Coexpression of recessive G701D and S773P mutants in polarized cells showed that these proteins could interact, yet no G701D mutant was detected at the basolateral membrane. Therefore, compound heterozygous patients expressing both recessive mutants (G701D/S773P) likely developed dRTA due to the lack of a functional kAE1 at the basolateral surface of a-intercalated cells.

BackgroundOpioid agonist drugs produce analgesia. However, long-term exposure to opioid agonists may lead to opioid dependence. The analgesic and addictive properties of opioid agonist drugs are mediated primarily via the mu-opioid receptor (MOR). Opioid agonists appear to alter neuronal morphology in key brain regions implicated in the development of opioid dependence. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid dependence.ResultsGPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was identified as a MORIP in a modified split ubiquitin yeast two-hybrid screen. GPR177 is an evolutionarily conserved protein that plays a critical role in mediating Wnt protein secretion from Wnt producing cells. The MOR/GPR177 interaction was validated in pulldown, coimmunoprecipitation, and colocalization studies using mammalian tissue culture cells. The interaction was also observed in rodent brain, where MOR and GPR177 were coexpressed in close spatial proximity within striatal neurons. At the cellular level, morphine treatment caused a shift in the distribution of GPR177 from cytosol to the cell surface, leading to enhanced MOR/GPR177 complex formation at the cell periphery and the inhibition of Wnt protein secretion.ConclusionsIt is known that chronic morphine treatment decreases dendritic arborization and hippocampal neurogenesis, and Wnt proteins are essential for these processes. We therefore propose that the morphine-mediated MOR/GPR177 interaction may result in decreased Wnt secretion in the CNS, resulting in atrophy of dendritic arbors and decreased neurogenesis. Our results demonstrate a previously unrecognized role for GPR177 in regulating cellular response to opioid drugs.