Defining the subcellular distribution and metabolic channeling of phosphatidylinositol.

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

Integrated regulation of the phosphatidylinositol cycle and phosphoinositide-driven lipid transport at ER-PM contact sites.

Among the structural phospholipids that form the bulk of eukaryotic cell membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the common precursor for low-abundance regulatory lipids, collectively referred to as polyphosphoinositides (PPIn). The metabolic turnover of PPIn species has received immense attention because of the essential functions of these lipids as universal regulators of membrane biology and their dysregulation in numerous human pathologies. The diverse functions of PPIn lipids occur, in part, by orchestrating the spatial organization and conformational dynamics of peripheral or integral membrane proteins within defined subcellular compartments. The emerging role of stable contact sites between adjacent membranes as specialized platforms for the coordinate control of ion exchange, cytoskeletal dynamics, and lipid transport has also revealed important new roles for PPIn species. In this review, we highlight the importance of membrane contact sites formed between the endoplasmic reticulum (ER) and plasma membrane (PM) for the integrated regulation of PPIn metabolism within the PM. Special emphasis will be placed on non-vesicular lipid transport during control of the PtdIns biosynthetic cycle as well as toward balancing the turnover of the signaling PPIn species that define PM identity.

Correction to: Polyphosphoinositide-Binding Domains: Insights from Peripheral Membrane and Lipid-Transfer Proteins.

This chapter was inadvertently published with an incorrect copyright holder. It has now been updated as below.

Polyphosphoinositide-Binding Domains: Insights from Peripheral Membrane and Lipid-Transfer Proteins.

Within eukaryotic cells, biochemical reactions need to be organized on the surface of membrane compartments that use distinct lipid constituents to dynamically modulate the functions of integral proteins or influence the selective recruitment of peripheral membrane effectors. As a result of these complex interactions, a variety of human pathologies can be traced back to improper communication between proteins and membrane surfaces; either due to mutations that directly alter protein structure or as a result of changes in membrane lipid composition. Among the known structural lipids found in cellular membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the membrane-anchored precursor of low-abundance regulatory lipids, the polyphosphoinositides (PPIn), which have restricted distributions within specific subcellular compartments. The ability of PPIn lipids to function as signaling platforms relies on both non-specific electrostatic interactions and the selective stereospecific recognition of PPIn headgroups by specialized protein folds. In this chapter, we will attempt to summarize the structural diversity of modular PPIn-interacting domains that facilitate the reversible recruitment and conformational regulation of peripheral membrane proteins. Outside of protein folds capable of capturing PPIn headgroups at the membrane interface, recent studies detailing the selective binding and bilayer extraction of PPIn species by unique functional domains within specific families of lipid-transfer proteins will also be highlighted. Overall, this overview will help to outline the fundamental physiochemical mechanisms that facilitate localized interactions between PPIn lipids and the wide-variety of PPIn-binding proteins that are essential for the coordinate regulation of cellular metabolism and membrane dynamics.

Comparative aspects of GnRH-Stimulated signal transduction in the vertebrate pituitary - Contributions from teleost model systems.

Gonadotropin-releasing hormone (GnRH) is a major regulator of reproduction through actions on pituitary gonadotropin release and synthesis. Although it is often thought that pituitary cells are exposed to only one GnRH, multiple GnRH forms are delivered to the pituitary of teleost fishes; interestingly this can include the cGnRH-II form usually thought to be non-hypophysiotropic. GnRHs can regulate other pituitary cell-types, both directly as well as indirectly, and multiple GnRH receptors (GnRHRs) may also be expressed in the pituitary, and even within a single pituitary cell-type. Literature on the differential actions of native GnRH isoforms in primary pituitary cells is largely derived from teleost fishes. This review will outline the diversity and complexity of GnRH-GnRHR signal transduction found within vertebrate gonadotropes as well as extra-gonadotropic sites with special emphasis on comparative studies from fish models. The implications that GnRHR transduction mechanisms are GnRH isoform-, function-, and cell-specific are also discussed.

Ligand-Biased Regulation of PtdIns(3,4,5)P3-Dependent Signal Transduction in GPCR Control of Pituitary Hormone Release.

Biased signaling describes the selective activation of signal transduction cascades by structurally related ligands downstream of shared G protein-coupled receptors (GPCRs). Although class I phosphoinositide 3-kinases (PI3Ks) are important components of GPCR-controlled transduction networks, little is known regarding the potential for biased regulation of class I PI3K-dependent signaling. The full complement of class I PI3K catalytic subunits (p110α, p110ß, p110δ, and p110γ) first appears in bony fishes and, despite being associated with distinct cellular functions, all class I PI3Ks produce the lipid second-messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. We have previously shown that 2 endogenous gonadotropin-releasing hormones (GnRH2 and GnRH3), which both signal through shared Gαq/11-coupled receptors, selectively activate different subsets of class I PI3K isoforms in their control of hormone release from goldfish (Carassius auratus) pituitary cells. Here, we tested the hypothesis that the biased activation of class I PI3K isoforms results in the selective recruitment of PtdIns(3,4,5)P3-sensitive effectors downstream of GnRH-stabilized GPCRs using pharmacological mapping. Our results reveal that distinct PtdIns(3,4,5)P3-sensitive effectors are involved in the differential control of GnRH2- and GnRH3-stimulated, as well as basal, hormone release and implicate the participation of noncanonical PtdIns(3,4,5)P3-sensitive transduction elements. Furthermore, observations using a selective inhibitor of the shared Gßγ-effector interaction surface indicate a role for Gßγ-dependent signaling in the integrated control of pituitary hormone exocytosis. These findings add to our understanding of functional selectivity in GPCR signal transduction networks, in general, and reveal the complexity of biased signaling downstream of class I PI3K catalytic activity.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110ß, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110ß, p110γ, and p110δ). Importantly, p110ß and p110γ, but not p110α or p110δ, can be directly activated by the Gßγ heterodimer of Gαßγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110ß, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110ß only regulate basal LH secretion. However, p110ß and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110ß and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gßγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

PI3K signalling in GnRH actions on dispersed goldfish pituitary cells: relationship with PKC-mediated LH and GH release and regulation of long-term effects on secretion and total cellular hormone availability.

Goldfish pituitary cells are exposed to two GnRHs, salmon (s)GnRH and chicken (c)GnRH-II. Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) both participate in acute sGnRH- and cGnRH-II-stimulated LH and GH release. Using goldfish pituitary cells, we examined the relationship between PI3K and PKC in acute LH and GH secretion, and PI3K involvement in chronic hormone release and total LH and GH availability. The PI3K inhibitor LY294002 did not affect PKC agonists-induced LH or GH release, and PKC agonists did not alter PI3K p85 phosphorylation, suggesting PKC activation is not upstream of PI3K in acute hormone release. In 2, 6, 12 and 24h treatments, LY294002 did not affect LH release but stimulated total LH availability at 6h. sGnRH stimulatory actions on LH release and total availability at 12 and 24h, and cGnRH-II effects on these parameters at 6h were inhibited by LY294002. LY294002 enhanced basal GH release at 2 and 6h, but reduced total GH at 12 and 24h. Increased GH release was seen following 6, 12 and 24h of sGnRH, and 2, 6 and 24h of cGnRH-II treatment but total GH availability was only elevated by 24h cGnRH-II treatment. Whereas LY294002 inhibited GH release responses to sGnRH at 12h and cGnRH-II at 6h, it attenuated cGnRH-II-elicited, but not sGnRH-induced, effects on total GH. These results indicate that PI3K differentially modulates long-term basal and GnRH-stimulated hormone release, and total hormone availability, in a time-, cell-type-, and GnRH isoform-selective manner.

Induction of phagocytosis and intracellular signaling by an inhibitory channel catfish leukocyte immune-type receptor: evidence for immunoregulatory receptor functional plasticity in teleosts.

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

MEK1/2 differentially participates in GnRH actions on goldfish LH and GH secretion and hormone protein availability: acute and long-term effects, in vitro.

Two endogenous gonadotropin-releasing hormones (GnRHs), sGnRH and cGnRH-II, stimulate LH and GH release via protein kinase C (PKC) signaling in goldfish. In this study, extracellular signal-regulated kinase kinase 1 and 2 (MEK1/2) involvement in acute and prolonged GnRH effects on goldfish gonadotrope and somatotrope functions, as well as potential interactions with PKC in the control of LH and GH release from goldfish pituitary cells was investigated. MEK1/2 inhibitors U0126 and PD098059 significantly decreased sGnRH but not cGnRH-II-stimulated GH release from perifused goldfish pituitary cells and U0126 significantly reduced the GH, but not the LH, release responses to synthetic PKC activators. In long-term static incubations (up to 24h) with goldfish pituitary cells, U0126 generally did not affect basal LH release but attenuated sGnRH- and cGnRH-II-induced LH release, as well as the time-dependent effects of sGnRH and/or cGnRH-II to elevate total LH availability (sum of release and cell content). sGnRH and cGnRH-II reduced cellular GH content and/or total GH availability at 2, 6, and 12h while static incubation with U0126 alone generally increased basal GH release but reduced cellular GH content and/or the total amount of GH available. U0126 also selectively reduced the sGnRH-induced GH release responses at 6 and 24h but paradoxically inhibited cGnRH-II-stimulated GH secretion while enhancing sGnRH-elicited GH release at 2h. Taken together, this study reveals the complexity of GnRH-stimulated MEK1/2 signaling and adds to our understanding of cell-type- and GnRH-isoform-selective signal transduction in the regulation of pituitary cell hormone release and production.

Calcium and other signalling pathways in neuroendocrine regulation of somatotroph functions.

Relative to mammals, the neuroendocrine control of pituitary growth hormone (GH) secretion and synthesis in teleost fish involves numerous stimulatory and inhibitory regulators, many of which are delivered to the somatotrophs via direct innervation. Among teleosts, how multifactorial regulation of somatotroph functions are mediated at the level of post-receptor signalling is best characterized in goldfish. Supplemented with recent findings, this review focuses on the known intracellular signal transduction mechanisms mediating the ligand- and function-specific actions in multifactorial control of GH release and synthesis, as well as basal GH secretion, in goldfish somatotrophs. These include membrane voltage-sensitive ion channels, Na(+)/H(+) antiport, Ca(2+) signalling, multiple pharmacologically distinct intracellular Ca(2+) stores, cAMP/PKA, PKC, nitric oxide, cGMP, MEK/ERK and PI3K. Signalling pathways mediating the major neuroendocrine regulators of mammalian somatotrophs, as well as those in other major teleost study model systems are also briefly highlighted. Interestingly, unlike mammals, spontaneous action potential firings are not observed in goldfish somatotrophs in culture. Furthermore, three goldfish brain somatostatin forms directly affect pituitary GH secretion via ligand-specific actions on membrane ion channels and intracellular Ca(2+) levels, as well as exert isoform-specific action on basal and stimulated GH mRNA expression, suggesting the importance of somatostatins other than somatostatin-14.

Differential involvement of phosphoinositide 3-kinase in gonadotrophin-releasing hormone actions in gonadotrophs and somatotrophs of goldfish, Carassius auratus.

In goldfish, two endogenous gonadotrophin-releasing hormones (GnRHs) [salmon (s)GnRH and chicken (c)GnRH-II] control maturational gonadotrophin-II [lutenising hormone (LH)] and growth hormone (GH) secretion via Ca(2+)-dependent intracellular signalling pathways. We investigated the involvement of phosphoinositide 3-kinase (PI3K) in GnRH-evoked LH and GH release and associated intracellular Ca(2+) increases ([Ca(2+)](i) ) in goldfish gonadotrophs and somatotrophs. Immunoreactive PI3K p85α, the predominant regulatory subunit for class IA PI3Ks, was detected in goldfish pituitary tissue extracts and both endogenous GnRH isoforms increased phosphorylation of PI3K p85α in excised pituitary fragments. sGnRH- and cGnRH-II-elicited LH release responses from primary cultures of pituitary cells and [Ca(2+)](i) increases in identified gonadotrophs were significantly reduced in the presence of PI3K inhibitors wortmannin (100 nm) and LY294002 (10 µm). Unexpectedly, wortmannin and LY294002 inhibited GnRH-evoked GH release but only attenuated the [Ca(2+)](i) response in identified somatotrophs to cGnRH-II, and not sGnRH. On the other hand, Ca(2+) ionophore-evoked LH and GH secretion remained unaltered in the presence of the PI3K inhibitors, suggesting that general decreases in the releasable hormone pool or sensitivity to [Ca(2+)](i) changes did not underlie the ability of wortmannin and LY294002 to reduce the actions of GnRH. These results provide the first evidence for the presence and involvement of PI3K in GnRH-induced LH and GH release in any primary pituitary cell system. In gonadotrophs, the inhibitory action of PI3K on both sGnRH and cGnRH-II involves the attenuation of their evoked [Ca(2+)](i); in contrast, GnRH isoform-specific effects occur in somatotrophs.

MicroRNA-193b represses cell proliferation and regulates cyclin D1 in melanoma.

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by > or = 50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3'untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development.