Receptor Activation Regulates Cortical, but not Vesicular Localization of NDP Kinase

We used immunofluorescence techniques to determine the localization of nucleoside diphosphate (NDP) kinase in NIH-3T3 fibroblasts. We found that cytoplasmic NDP kinase can be separated into two populations according to subcellular localization and response to extracellular stimuli. Specifically, within minutes of stimulation of resting fibroblasts with serum, growth factors or bombesin, a portion of NDP kinase becomes associated with membrane ruffles and lamellipodia. Another pool of NDP kinase accumulates independently of stimulation around intracellular vesicles. Transfection of cells with activated Rac mimics, whereas expression of dominant negative Rac inhibits, the effects of extracellular stimulation on the translocation of NDP kinase to the cell cortex. Neither Rac mutant affects the vesicle-associated pool. Association of NDP kinase with vesicles depends on microtubule integrity and is disrupted by nocodazole. In cell-free assays NDP kinase binds tightly to membrane vesicles associated with taxol-stabilized microtubules. Binding of NDP kinase to this fraction is reduced by ATP and abolished by GTP, as well as guanine nucleotides that are NDP kinase substrates. Thus, the localization of the two NDP kinase pools identified here is regulated independently by distinct cellular components: the appearance of cortical NDP kinase is a consequence of Rac activation, whereas vesicular NDP kinase is responsive to microtubule dynamics and nucleotides, in particular GTP. These results suggest that in fibroblasts NDP kinase participates in Racrelated cortical events and in GTP-dependent processes linked to intracellular vesicle trafficking

Association of Nucleoside Diphosphate Kinase with Microtubule-Based Structures

Cytosolic nucleoside diphosphate kinases (NDPKs) have been implicated in a variety of signaling pathways that occur at membranes, including those that control cell migration and spreading. This is particularly intriguing, as cytosolic NDPKs (NDPK A and NDPK B) are soluble proteins and do not have membrane-binding motifs, leading to the question: how do NDPK\u27s participate in such a wide array of membrane signaling processes? Our lab has shown that one portion of cytosolic NDPK is translocated to the ruffling plasma membrane upon activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) and that the Rac1 signaling pathway is responsible for that migration. Although NDPK does not bind directly to Rac1, it moves to the cell periphery in conjunction with Rac1. While investigating the association of cytosolic NDPK with the plasma membrane, we found that another pool of NDPK is bound to membrane vesicles that are associated with microtubules (Mt/Ves). A detailed study of this NDPK population shows that, unlike the pool that is involved in Rac1 signaling, NDPK\u27s presence in these vesicles is not dependent on extracellular stimulation; rather, it is controlled by the nucleotide triphosphate to nucleotide diphosphate ratio ([NTP]/[NDP]), as evidenced by the effect of nucleotides on Mt/Ves isolated from fibroblasts. More importantly, purified and cytosolic NDPKs bind to both immobilized lipids and liposomes in a nucleotide-sensitive manner. This indicates that NDPK can bind directly to intracellular membrane compartments, most likely to provide CTP for phospholipid biosynthesis and GTP for the many small GTPases involved in microtubule-dependent traffic. We also found that NDPK localizes to yet another microtubule-based cell compartment: the sensory primary cilium, an organelle implicated in many signaling pathways. NDPK enters the cilium during its development, when it reaches about 5.5 microns (or 24% of final primary cilia length) in A6 cells. In primary cilia NDPK is present in the soluble portion, or matrix, and in association with the membrane fraction. The function of NDPK within primary cilia is most likely to regenerate GTP for microtubule turnover and for signaling systems, making it an important contributor to primary cilia structure and function

Modulation of Kir6.1 channels heterologously expressed in HEK-293 cells by nicotine and acetylocholine

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied. Non-transfected HEK-293 cells possess an endogenous K+ current with current density of –3.2 ± 1.4 pA/pF at –150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at –150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6,

Cellular mechanisms that regulate the endogenous mono-ADP-ribosylation of the G protein βγ subunit.

Mono-ADP-ribosylation is a reversible, post-translational modification of cellular proteins that has been implicated in regulation of signal transduction, muscle cell differentiation, and protein trafficking and secretion. The reaction is catalysed by mono-ADP-ribosyltransferases that transfer a single ADP-ribose moiety from p-NAD+ to a specific amino-acid of acceptor proteins. An ADP-ribosylation reaction occurs in intact cells on the p subunit of heterotrimeric G proteins that is carried out by an arginine- specific, plasma-membrane-associated, mono-ADP-ribosyltransferase. This modification is reversed by a cytosolic ADP-ribosylhydrolase that regenerates native Py dimer by releasing the bound ADP-ribose. Once ADP-ribosylated, the py dimer is inactive towards its effector enzymes, such as adenylyl cyclase, phosphoinositide 3-kinase and phospholipase C. It thus appears that endogenous P subunit mono-ADP-ribosylation might represent a novel cellular mechanism for the modulation of the G-protein-mediated signal transduction machinery through a direct regulation of the py dimer. In this study, the mechanisms that regulate endogenous mono-ADP-ribosylation of the p subunit have been investigated. The reaction appears to be under hormonal control both in vitro and in vivo, since the levels of ADP-ribosylated p are increased upon activation of certain G-protein-coupled receptors (GPCRs), such as thrombin, serotonin and cholecystokinin receptors. Conversely, hormonal stimulation by additional GPCRs, such as the GnRH receptor, can lead to a decrease in p subunit mono-ADP-ribosylation. Thus, ADP-ribosylation of the py dimer can be differentially regulated by different GPCRs in a receptor-type-dependent manner. In addition, the involvement of the ADP-ribosylating factor ARF6 in GnRH-mediated regulation of p subunit mono-ADP-ribosylation is demonstrated. Indeed, removal of ARF6 from plasma membranes results in loss of GnRH-mediated inhibition of p subunit mono-ADP-ribosylation, which can be fully restored by re-addition of purified ARF6. In conclusion, the results reported in this thesis allow the definition of the mechanisms that regulated endogenous ADP-ribosylation of the p subunit, and demonstrate a novel role for ARF6 in hormonal regulation of p subunit mono-ADP-ribosylation

Characterization and functional analysis of the P2Y₂R gene promoter

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on April 21, 2009)Includes bibliographical references.Thesis (M.S.) University of Missouri-Columbia 2006.Dissertations, Academic -- University of Missouri--Columbia -- Biochemistry (Agriculture)Extracellular nucleotides can bind to the P2Y₂R and modulate proliferation and migration of smooth muscle cells, which is known to be involved in intimal hyperplasia that accompanies atherosclerosis and post-angioplasty restenosis. Moreover, the P2Y₂R is upregulated in vascular smooth muscle cells and endothelial cells in response to tissue injury. These findings suggest that the P2Y₂R is a potential target for the pharmacological control of progression of atherosclerosis and post-angioplasty restenosis. However, the mechanisms governing P2Y₂R up-regulation remain unknown. In this study, we have cloned a 2071 bp 5'-flanking region of the P2Y₂R gene in a reporter vector and carried out a serial deletion analysis. The deletion of a 175 bp region completely abolished promoter function and results further indicate that the P2Y₂R gene promoter uses an array of positive and negative response elements in the regulation of gene expression. Furthermore, other results show that the cytokine IL-1[beta] may be involved in down-regulation of P2Y₂R activity in human coronary artery endothelial cells. Further studies will potentially lead to the identification of novel pathways involved in the regulation of P2Y₂R gene expression, information that might be useful to suppress neointimal hyperplasia in atherosclerosis and the restenosis of angioplasty

Developing a recombinant model of the P2Y1 and P2Y11 receptor interactions mediating relaxation in gut smooth muscle

ATP and ADP mediate gut smooth muscle relaxation through two receptors, P2Y1 and P2Y11. This project aims to investigate the interaction between these two receptors by developing a recombinant model of the P2Y receptors expressed in gut smooth muscle cells (SMCs) by transfecting the human P2Y11 receptor cDNA into CHO-K1 cells, which express an endogenous P2Y1 receptor. Individual clonal cell lines expressing different densities of hP2Y11 were isolated from this stably-transfected CHO-K1:P2Y11 pool and characterized. A clone expressing a “high” density of hP2Y11 (13) and a clone expressing a “low” density of hP2Y11 (6) were selected for further study. Control 1321N1 cell lines expressing each receptor in isolation (1321N1-hP2Y1 and 1321N1-hP2Y11) were used for comparison purposes. The potency (EC50) of eight different nucleotide agonists was determined in calcium assays in the co-expressing cell lines. ADP and 2meSATP responses were biphasic in clone 13 but monophasic in clone 6. To investigate the nature of the two sites of the biphasic curves in clone 13, the effect of MRS 2179, NF 340 and Reactive Red on agonist responses was determined. MRS 2179 antagonized the high affinity site of the biphasic ADP and 2meSATP responses in clone 13 without affecting the low affinity site. NF 340 had no effect on agonist responses in clone 13. Reactive Red antagonized both sites of the biphasic curves in clone 13. These data suggest that the high-affinity site of the biphasic ADP and 2meSATP responses in clone 13 corresponds to P2Y1. The low-affinity site of the 2meSATP curve is most likely P2Y11. The low-affinity site the ADP response displays both P2Y1 and P2Y11-like. The novel ADP site, therefore, is elicited by differences in the expression level of P2Y11 and may correspond to a P2Y1:hP2Y11 receptor heteromer or a macromolecular complex containing both P2Y1 and P2Y11

Multiple domain 'nexus' proteins in receptor-mediated cell signaling

Signal transduction is the fundamental biological process of converting changes in extracellular information into changes in intracellular functions. It controls a wide range of cellular activities, from the release of neurotransmitters and hormones, to integrated cellular decisions of proliferation, differentiation, survival, or death. The vast majority of extracellular signaling molecules exert their cellular effects through activation of G protein-coupled receptors (GPCRs); however, the Gprotein coupled paradigm is by no means the exclusive mechanism of membrane receptor signal transduction. Polypeptide ligands such as nerve growth factor act exclusively on receptor tyrosine kinase receptors (RTKs) to promote signaling. GPCRs and RTKs both form an interface between extracellular and intracellular physiology by converting hormonal signals into changes in intracellular metabolism and ultimately cell phenotype. Initially, it was thought that GPCRs and RTKs represented linear and distinct signaling pathways that converge on downstream targets to regulate cell division and gene transcription. However, activation of second messenger generating systems do not fully explain the range of effects of GPCR or RTK activation on biological processes such as differentiation and cell growth. Recent work has revealed that GPCR and RTK signaling pathways are not mutually exclusive of one another; in fact, they often function as partners, forming complex signaling networks through scaffold/nexus proteins. The work described herein examines the complexity of signal regulation by multifunctional nexus proteins. I showed that the activation of phospholipase C-ε by Gα12/13-coupled receptors occurs through a mechanism involving the small GTPase Rho. I demonstrated the usefulness and complexities of ‘regulators of G-protein signaling’ (RGS proteins) for discerning the Gα selectivity of GPCR signaling. Finally, I found that RGS12, in addition to regulating Gα signaling, acts as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation. The work presented here expands our understanding of how multiple domain proteins facilitate convergence and cross-regulation of RTK, heterotrimeric G-protein, and Rassuperfamily signaling