Arieh A. Katz

A novel mammalian receptor for the evolutionarily conserved type II GnRH.

Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K+ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to Gαq/11 and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.

G protein βγ complex-mediated apoptosis by familial Alzheimer's disease mutant of APP

In familial Alzheimers disease (FAD), three missense mutations, V642I, V642F and V642G, that co‐segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin‐sensitive apoptosis in an FAD trait‐linked manner by activating the G protein Go, which consists of Gαo and Gβγ subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated Gαo or Gαi induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy‐terminal amino acids 495–689 of the β‐adrenergic receptor kinase‐1, which blocks the specific functions of Gβγ. Co‐transfection of Gβ2γ2 cDNAs, but not that of other Gβxγz (x = 1–3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl‐2. These data implicate Gβγ as a cell death mediator for the FAD‐associated mutant of APP.

Retention and Silencing of Prepro-GnRH-II and Type II GnRH Receptor Genes in Mammals

The decapeptide hypothalamic-pituitary gonadotrophin-releasing hormone (GnRH)-I and the type I GnRH receptor drive the reproductive hormonal cascade in mammals by stimulating synthesis and secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH). Mammals possess a second GnRH system composed of a related hormone, GnRH-II (differing from GnRH-I by three amino acid residues), and the type II GnRH receptor. In many mammalian species, one or both of the GnRH-II system genes are disrupted or deleted, rendering their products non-functional. This includes humans who possess a gene encoding GnRH-II but lack a functional type II GnRH receptor. Here we examined the genes encoding prepro-GnRH-II (GnRH2) and the type II GnRH receptor (GnRHR2) in more than 20 mammalian species, encompassing 10 orders, to determine whether they encode functional proteins. The structural organisation of both genes in most mammalian genome sequence assemblies was poorly annotated or incompletely described. Our findings show significant variation in the DNA sequence conservation and functional status of each gene, even between closely related species. Prepro-GnRH-II was functionally compromised in 12/22 species and the type II GnRH receptor gene was disrupted in 14/22 species. Retention of large sections of each gene in most mammalian genomes suggests that mammalian ancestors had a functional GnRH-II system. Gene disruptions were due to a spectrum of mutations which must have occurred independently after the evolutionary divergence of mammals from ancestral animals. The genetic information will be useful for understanding the physiological role of the GnRH-II system and establishing animal models for functional studies.

Dynamic reciprocity: the role of annexin A2 in tissue integrity

Interactions between cells and the extracellular matrix are integral to tissue development, remodelling and pathogenesis. This is underlined by bi-directional flow of information signalling, referred to as dynamic reciprocity. Annexin A2 is a complex and multifunctional protein that belongs to a large family of Ca2+-dependent anionic phospholipid and membrane-binding proteins. It has been implicated in diverse cellular processes at the nuclear, cytoplasmic and extracellular compartments including Ca2+-dependent regulation of endocytosis and exocytosis, focal adhesion dynamics, transcription and translation, cell proliferation, oxidative stress and apoptosis. Most of these functions are mediated by the annexin A2-S100A10 heterotetramer (AIIt) via its ability to simultaneously interact with cytoskeletal, membrane and extracellular matrix components, thereby mediating regulatory effects of extracellular matrix adhesion on cell behaviour and vice versa. While Src kinase-mediated phosphorylation of filamentous actin-bound AIIt results in membrane-cytoskeletal remodelling events which control cell polarity, cell morphology and cell migration, AIIt at the cell surface can bind to a number of extracellular matrix proteins and catalyse the activation of serine and cysteine proteases which are important in facilitating tissue remodelling during tissue repair, neoangiogenesis and pathological situations. This review will focus on the role of annexin A2 in regulating tissue integrity through intercellular and cell-extracellular matrix interaction. Annexin A2 is differentially expressed in various tissue types as well as in many pathologies, particularly in several types of cancer. These together suggest that annexin A2 acts as a central player during dynamic reciprocity in tissue homeostasis.

Ion dynamics during seizures

Changes in membrane voltage brought about by ion fluxes through voltage and transmitter-gated channels represent the basis of neural activity. As such, electrochemical gradients across the membrane determine the direction and driving force for the flow of ions and are therefore crucial in setting the properties of synaptic transmission and signal propagation. Ion concentration gradients are established by a variety of mechanisms, including specialized transporter proteins. However, transmembrane gradients can be affected by ionic fluxes through channels during periods of elevated neural activity, which in turn are predicted to influence the properties of on-going synaptic transmission. Such activity-induced changes to ion concentration gradients are a feature of both physiological and pathological neural processes. An epileptic seizure is an example of severely perturbed neural activity, which is accompanied by pronounced changes in intracellular and extracellular ion concentrations. Appreciating the factors that contribute to these ion dynamics is critical if we are to understand how a seizure event evolves and is sustained and terminated by neural tissue. Indeed, this issue is of significant clinical importance as status epilepticus—a type of seizure that does not stop of its own accord—is a life-threatening medical emergency. In this review we explore how the transmembrane concentration gradient of the six major ions (K+, Na+, Cl−, Ca2+, H+and HCO3−) is altered during an epileptic seizure. We will first examine each ion individually, before describing how multiple interacting mechanisms between ions might contribute to concentration changes and whether these act to prolong or terminate epileptic activity. In doing so, we will consider how the availability of experimental techniques has both advanced and restricted our ability to study these phenomena.

Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection.

Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells

The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.

Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo.

Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A(VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.

Pro7.33(303) of the human GnRH receptor regulates selective binding of mammalian GnRH.

Mammalian gonadotropin releasing hormone (GnRH) receptors have a conserved acidic residue (Glu7.32(301) or Asp7.32(302)) in extracellular loop (ECL) three that confers selectivity for mammalian GnRH, which has Arg8. Comparison of mammalian and non-mammalian GnRH receptors suggested that the acidic residue is not the only determinant of ligand selectivity in mammalian receptors. The acidic residue is followed by a conserved Pro7.33 in mammalian GnRH receptors, but not non-mammalian receptors. Unique structural constraints imposed by Pro residues suggested that Pro7.33 determines selective binding of Arg8-containing GnRH, by stabilising the conformation of the third extracellular loop of the receptor. Substituting Pro7.33(303) or introducing Pro to position 7.31 decreased affinity for GnRH, but not analogs lacking Arg8. Substituting Pro7.33(303) changed the predicted alpha-helix content of the loop-helix interface. These results show that Pro7.33(303) of the human GnRH receptor is required for selective high affinity binding of mammalian GnRH and supports the hypothesis that Pro7.33(303) stabilises a loop conformation that is necessary for selective ligand binding.