Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation.

We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs.

Expression of GPR177 (Wntless/Evi/Sprinter), a highly conserved Wnt-transport protein, in rat tissues, zebrafish embryos, and cultured human cells.

GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse, and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GPR177 polypeptides are expressed throughout embryogenesis, and are detectable as early as 1 hr post-fertilization. In situ hybridization analysis reveals that gpr177 mRNA expression is prominent in embryonic zebrafish brain and ear. Structural studies suggest that GPR177 is modified by N-linked sugars, and that the protein contains an even number of transmembrane segments. The relatively ubiquitous expression of GPR177 suggests that this protein may serve to regulate Wnt secretion in a variety of embryonic and adult tissue types.

Developmental expression of doublesex-related transcripts in the common house spider, Parasteatoda tepidariorum.

Sex determination is a rapidly evolving biological process controlled by differential gene expression. One family of transcription factors that initiate sex-specific gene expression and differentiation in many animal species are the Doublesex and Mab-3 (DM) domain proteins. While much is known about Doublesex-related proteins in various insect orders and commonly studied model systems, little is known about their function in basally branching arthropods. Spiders are an emerging model for molecular and evolutionary development that could fill this gap. Arachnids share an ancient whole-genome duplication providing a unique opportunity to study the effect of major genomic rearrangements on the evolution of developmental processes. In this study, we aimed to identify the repertoire of Dsx-related proteins encoded by the genome of the common house spider, Parasteatoda tepidariorum. While insects have four DM domain proteins, the P. tepidariorum genome encodes seven, indicating the possibility of duplicate retention. At least four of the DM protein genes demonstrated sex bias expression in adult spiders. Embryonic expression of these genes suggests roles in development of the spinnerets, nervous system, and appendages.

Identification of zebrafish A2 adenosine receptors and expression in developing embryos.

The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson's disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62-74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.

Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish.

BACKGROUND: The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated. RESULTS: We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4kbeta, dan, and pink1 in semicircular canal formation. CONCLUSION: Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kbeta/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.

Alternative splicing of the Wnt trafficking protein, Wntless and its effects on protein-protein interactions.

BACKGROUND: Wntless (Wls) is a protein that regulates secretion of Wnt signaling molecules from Wnt-producing cells. Wnt signaling is known to be critical for several developmental and homeostatic processes. However, Wnt-independent functions of Wls are now being elucidated. Primates express an alternative splice variant of Wls (here termed WlsX). WlsX contains an alternatively spliced COOH-terminus, and does not appear to be able to sustain significant levels of WNT secretion because of its inability to undergo retrograde trafficking to the endoplasmic reticulum. The functional significance for this alternatively spliced form of Wls has not yet been elucidated. We previously identified a cohort of Wls interacting proteins using a combination of yeast 2-hybrid and candidate gene approaches. RESULTS: In the present study, we analyzed the interaction of WlsX with previously identified Wls interactors, and additionally screened for novel protein interactors of WlsX utilizing a membrane yeast two hybrid screen. Three novel Wls interactors, Glycoprotein M6A (GPM6A), Alkylglycerol Monooxygenase (AGMO), and ORAI1 were identified. Each of these novel WlsX interactors, as well as all other Wls interacting proteins identified previously, with the exception of the mu-opioid receptor, were found to interact with both Wls and WlsX splice forms. We show that WlsX can form homodimers, but that WlsX may not interact with Wls. CONCLUSIONS: WlsX has the ability to form homodimers and to interact with most known Wls interacting proteins. Taken together, our results suggest that Wls and WlsX may have overlapping, but distinct functions, including sensitivity to opioid drugs. While studies have focused on the ability of Wls interacting proteins to affect Wnt secretion, future efforts will explore the reciprocal regulation of these proteins by Wls, possibly via Wnt-independent mechanisms.

Identifying novel members of the Wntless interactome through genetic and candidate gene approaches.

Wnt signaling is an important pathway that regulates several aspects of embryogenesis, stem cell maintenance, and neural connectivity. We have recently determined that opioids decrease Wnt secretion, presumably by inhibiting the recycling of the Wnt trafficking protein Wntless (Wls). This effect appears to be mediated by protein-protein interaction between Wls and the mu-opioid receptor (MOR), the primary cellular target of opioid drugs. The goal of this study was to identify novel protein interactors of Wls that are expressed in the brain and may also play a role in reward or addiction. Using genetic and candidate gene approaches, we show that among a variety of protein, Wls interacts with the dopamine transporter (target of cocaine), cannabinoid receptors (target of THC), Adenosine A2A receptor (target of caffeine), and SGIP1 (endocytic regulator of cannabinoid receptors). Our study shows that aside from opioid receptors, Wntless interacts with additional proteins involved in reward and/or addiction. Future studies will determine whether Wntless and WNT signaling play a more universal role in these processes.

Early avoidance of a heroin-paired taste-cue and subsequent addiction-like behavior in rats.

The ability to predict individual vulnerability to substance abuse would allow for a better understanding of the progression of the disease and development of better methods for prevention and/or early intervention. Here we use drug-induced devaluation of a saccharin cue in an effort to predict later addiction-like behavior in a model akin to that used by Deroche-Gamonet et al. (2004) and seek to link such vulnerability to changes in expression of various mu opioid receptor and D2 receptor-interacting proteins in brain. The results show that the greatest heroin-induced suppression of intake of a saccharin cue is associated with the greatest vulnerability to later addiction-like behavior and to differences in the expression of WLS, ß-catenin, and NCS-1 in brain compared to rats that exhibited the least suppression of intake of the heroin-paired cue and/or saline controls. Finally, because the self-administration model employed produced no significant differences in drug intake between groups, overall, the resultant changes in protein expression can be more closely linked to individual differences in motivation for drug.

Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

Genomic strategies for the identification of dopamine receptor genes in zebrafish.

In this chapter, we describe the identification and cloning of D2-like dopamine receptor (DR) genes in zebrafish, a vertebrate model genetic organism. To identify DR genes, we performed searches of the zebrafish genomic sequence database that yielded contig segments of several D2-like DR genes. From these sequences, we amplified full-length cDNAs encoding three D2, one D3, and three D4 DR receptor subtypes via RT-PCR. The predicted proteins displayed 57-72% amino acid identity when compared to their human DR counterparts. To validate the identity of zebrafish DR genes, each of the genes was mapped by using the T51 radiation hybrid panel. With the exception of drd2b and drd4b, each of the zebrafish DR genes mapped to chromosomal positions that were syntenic with regions of human chromosomes containing orthologs of the zebrafish DR genes. To further validate the identity of the D2-like DR genes in zebrafish, we conducted phylogenetic analysis which supported the predicted identities of the cloned DR receptor cDNAs.

MOR is not enough: identification of novel mu-opioid receptor interacting proteins using traditional and modified membrane yeast two-hybrid screens.

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.

Otoc1: a novel otoconin-90 ortholog required for otolith mineralization in zebrafish.

Within the vestibular system of virtually all vertebrate species, gravity and linear acceleration are detected via coupling of calcified masses to the cilia of mechanosensory hair cells. The mammalian ear contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while otolith matrix protein (OMP) is the most abundant matrix protein found in the otoliths of teleost fish. We have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 was detected in the ear between 15 hpf and 72 hpf, and was restricted primarily to the macula and the developing epithelial pillars of the semicircular canals. Expression of otoc1 was also detected in epiphysis, optic stalk, midbrain, diencephalon, flexural organ, and spinal cord. During embryogenesis, expression of otoc1 mRNA preceded the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produced a variety of aberrant otolith phenotypes. Our results suggest that Otoc1 may serve to nucleate calcium carbonate mineralization of aragonitic otoliths.