Kenneth Watterson | University of Glasgow (original) (raw)

Papers by Kenneth Watterson

Research paper thumbnail of The regulation of the human A1 adenosine receptor and the sphingosine 1-phosphate receptor, EDG1 /

Ph. D. thesis submitted to the Institute of Biomedical and Life Sciences, University of Glasgow, ... more Ph. D. thesis submitted to the Institute of Biomedical and Life Sciences, University of Glasgow, 2002. Thesis (Ph. D.)--University of Glasgow, 2002. Includes bibliographical references.

Research paper thumbnail of Pleiotropic actions of sphingosine-1-phosphate

Progress in Lipid Research, 2003

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse cellu... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse cellular responses including, growth, survival, cytoskeleton rearrangements and movement. S1P plays an important role during development, particularly in vascular maturation and has been implicated in pathophysiology of cancer, wound healing, and atherosclerosis. This review summarizes the evidence showing that signaling induced by S1P is complex and involves both intracellular and extracellular actions. The intracellular effects of S1P remain speculative awaiting the identification of specific targets whereas the extracellular effects of S1P are clearly mediated through the activation of five specific G protein coupled receptors, called S1P 1À5 . Recent studies demonstrate that intracellular generated S1P can act in a paracrine or autocrine manner to activate its cell surface receptors. #

Research paper thumbnail of Regulation of fibroblast functions by lysophospholipid mediators: Potential roles in wound healing

Wound Repair and Regeneration, 2007

The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphat... more The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S1P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S1P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFb and S1P cross-talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.

Research paper thumbnail of Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The Journal of Cell Biology, 2004

T NGF-induced neurite extension was suppressed by downregulation of S1P 1 expression with antisen... more T NGF-induced neurite extension was suppressed by downregulation of S1P 1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P 1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P 2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Research paper thumbnail of Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells

The FASEB Journal, 2003

The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmati... more The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a G i -independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP 3 -sensitive calcium stores partially blocked increases in [Ca 2؉ ] i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.-Rosenfeldt, H. M., Amrani, Y., Watterson, K. R., Murthy, K. S., Panettieri, R. A., Jr., Spiegel, S. Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells. FASEB J. 17, 1789 -1799 (2003)

Research paper thumbnail of Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone

The FASEB Journal, 2007

Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth m... more Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P 1 and S1P 2 , only S1P 2 appeared to be involved in S1Pinduced contraction, since SEW2871 (S1P 1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P 2 ) were poor contractile agents. In agreement, the S1P 2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to -phosphate and the immunosuppressant, fty720-phosphate, regulate detrusor muscle tone. FASEB J. 21, 2818 -2828 (2007)

Research paper thumbnail of Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

Neurosignals, 2013

an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the la... more an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

Research paper thumbnail of Dual Regulation of EDG1/S1P1 Receptor Phosphorylation and Internalization by Protein Kinase C and G-protein-coupled Receptor Kinase 2

Journal of Biological Chemistry, 2002

Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor &a... more Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable.

Research paper thumbnail of Leptin-dependent Phosphorylation of PTEN Mediates Actin Restructuring and Activation of ATP-sensitive K+ Channels

Journal of Biological Chemistry, 2009

Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinas... more Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of

Research paper thumbnail of AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels

Journal of Bioenergetics and Biomembranes, 2013

Glucose-sensing (GS) behaviour in pancreatic βcells is dependent on ATP-sensitive K + channel (K ... more Glucose-sensing (GS) behaviour in pancreatic βcells is dependent on ATP-sensitive K + channel (K ATP ) activity, which is controlled by the relative levels of the K ATP ligands ATP and ADP, responsible for closing and opening K ATP , respectively. However, the mechanism by which β-cells transfer energy status from mitochondria to K ATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMPactivated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep K ATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP 5 A blocked this action of AMPK. Using conditions that maximised AMPK-induced K ATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that K ATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane K ATP by phosphotransfer cascades.

Research paper thumbnail of Dissecting the regulatory mechanisms controlling inhibitory adenosine receptor signaling

Drug Development Research, 2003

The ability of target cells to respond to rapid changes in extracellular adenosine concentrations... more The ability of target cells to respond to rapid changes in extracellular adenosine concentrations that occur in response to ischemia is determined by their complement of adenosine receptors. Four adenosine receptor subtypes, termed A 1 , A 2A , A 2B, and A 3 , have been identified by pharmacological and molecular cloning studies. In addition to displaying distinct pharmacological characteristics, each receptor mediates its intracellular effects by coupling to defined G-protein families. Our studies have concentrated on applying a combination of molecular biological, biochemical, and cell biological approaches to define the molecular mechanisms by which adenosine receptor-derived signaling events are regulated. These studies have revealed that the desensitization of closely related adenosine receptors, which bind the same agonist in vivo (i.e., adenosine) and activate the same family of G proteins, is mediated by unique receptor-specific mechanisms. In this review, the nature of these differences, and how they could potentially be exploited to test novel strategies for enhancing the cardioprotective effects of inhibitory adenosine receptor activation in vivo, will be described. Drug Dev. Res. 58:302-314, 2003.

Research paper thumbnail of The role of sphingosine-1-phosphate in smooth muscle contraction

Cellular Signalling, 2005

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is known to mediate div... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is known to mediate diverse cellular responses including cell growth, survival, and migration. Most of these effects have been attributed to its binding to a specific subfamily of G protein-coupled receptors (GPCR), namely S1P(1-5). Recent studies have suggested that S1P also plays a prominent role in the contraction of various types of smooth muscle. This review provides a brief overview of its role in this process and also highlights how S1P-dependent signaling serves as an important regulator of smooth muscle contraction.

Research paper thumbnail of Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors

Blood, 2005

Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting... more Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.

Research paper thumbnail of Subtype-Specific Regulation of Receptor Internalization and Recycling by the Carboxyl-Terminal Domains of the Human A 1 and Rat A 3 Adenosine Receptors:  Consequences for Agonist-Stimulated Translocation of Arrestin3 †

Biochemistry, 2002

In this study, we have characterized the differential effects on inhibitory adenosine receptor (A... more In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys 302,305 Ala-mutated rat A 3 AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A 1 AR (Cys 309 Ala) had no effect on receptor internalization. Moreover, unlike the WT A 3 AR, the entire pool of internalized mutant A 3 AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A 3 ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A 1 AR with the 14 GRK-phosphorylated amino acids of the rat A 3 AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A 1 CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A 1 AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys 302,305 Ala-mutated A 3 ARs, as well as the A 1 CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A 1 AR nor the rat A 3 AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory ARmediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A 3 AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.

Research paper thumbnail of The S1P2 Receptor Negatively Regulates Platelet-Derived Growth Factor-Induced Motility and Proliferation

Molecular and Cellular Biology, May 15, 2005

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specif... more Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G proteincoupled receptors, named S1P 1 to S1P 5 . In this study, we found that cross-communication between plateletderived growth factor receptor and S1P 2 serves as a negative damper of PDGF functions. Deletion of the S1P 2 receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P 1 and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P 2 deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P 2 -null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P 2 reduced DNA synthesis and expression of SphK1. Thus, S1P 2 serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.

Research paper thumbnail of The regulation of the human A1 adenosine receptor and the sphingosine 1-phosphate receptor, EDG1 /

Ph. D. thesis submitted to the Institute of Biomedical and Life Sciences, University of Glasgow, ... more Ph. D. thesis submitted to the Institute of Biomedical and Life Sciences, University of Glasgow, 2002. Thesis (Ph. D.)--University of Glasgow, 2002. Includes bibliographical references.

Research paper thumbnail of Pleiotropic actions of sphingosine-1-phosphate

Progress in Lipid Research, 2003

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse cellu... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse cellular responses including, growth, survival, cytoskeleton rearrangements and movement. S1P plays an important role during development, particularly in vascular maturation and has been implicated in pathophysiology of cancer, wound healing, and atherosclerosis. This review summarizes the evidence showing that signaling induced by S1P is complex and involves both intracellular and extracellular actions. The intracellular effects of S1P remain speculative awaiting the identification of specific targets whereas the extracellular effects of S1P are clearly mediated through the activation of five specific G protein coupled receptors, called S1P 1À5 . Recent studies demonstrate that intracellular generated S1P can act in a paracrine or autocrine manner to activate its cell surface receptors. #

Research paper thumbnail of Regulation of fibroblast functions by lysophospholipid mediators: Potential roles in wound healing

Wound Repair and Regeneration, 2007

The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphat... more The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S1P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S1P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFb and S1P cross-talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.

Research paper thumbnail of Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The Journal of Cell Biology, 2004

T NGF-induced neurite extension was suppressed by downregulation of S1P 1 expression with antisen... more T NGF-induced neurite extension was suppressed by downregulation of S1P 1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P 1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P 2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Research paper thumbnail of Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells

The FASEB Journal, 2003

The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmati... more The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a G i -independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP 3 -sensitive calcium stores partially blocked increases in [Ca 2؉ ] i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.-Rosenfeldt, H. M., Amrani, Y., Watterson, K. R., Murthy, K. S., Panettieri, R. A., Jr., Spiegel, S. Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells. FASEB J. 17, 1789 -1799 (2003)

Research paper thumbnail of Sphingosine-1-phosphate and the immunosuppressant, FTY720-phosphate, regulate detrusor muscle tone

The FASEB Journal, 2007

Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth m... more Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P 1 and S1P 2 , only S1P 2 appeared to be involved in S1Pinduced contraction, since SEW2871 (S1P 1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P 2 ) were poor contractile agents. In agreement, the S1P 2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to -phosphate and the immunosuppressant, fty720-phosphate, regulate detrusor muscle tone. FASEB J. 21, 2818 -2828 (2007)

Research paper thumbnail of Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

Neurosignals, 2013

an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the la... more an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

Research paper thumbnail of Dual Regulation of EDG1/S1P1 Receptor Phosphorylation and Internalization by Protein Kinase C and G-protein-coupled Receptor Kinase 2

Journal of Biological Chemistry, 2002

Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor &a... more Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable.

Research paper thumbnail of Leptin-dependent Phosphorylation of PTEN Mediates Actin Restructuring and Activation of ATP-sensitive K+ Channels

Journal of Biological Chemistry, 2009

Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinas... more Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of

Research paper thumbnail of AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels

Journal of Bioenergetics and Biomembranes, 2013

Glucose-sensing (GS) behaviour in pancreatic βcells is dependent on ATP-sensitive K + channel (K ... more Glucose-sensing (GS) behaviour in pancreatic βcells is dependent on ATP-sensitive K + channel (K ATP ) activity, which is controlled by the relative levels of the K ATP ligands ATP and ADP, responsible for closing and opening K ATP , respectively. However, the mechanism by which β-cells transfer energy status from mitochondria to K ATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMPactivated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep K ATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP 5 A blocked this action of AMPK. Using conditions that maximised AMPK-induced K ATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that K ATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane K ATP by phosphotransfer cascades.

Research paper thumbnail of Dissecting the regulatory mechanisms controlling inhibitory adenosine receptor signaling

Drug Development Research, 2003

The ability of target cells to respond to rapid changes in extracellular adenosine concentrations... more The ability of target cells to respond to rapid changes in extracellular adenosine concentrations that occur in response to ischemia is determined by their complement of adenosine receptors. Four adenosine receptor subtypes, termed A 1 , A 2A , A 2B, and A 3 , have been identified by pharmacological and molecular cloning studies. In addition to displaying distinct pharmacological characteristics, each receptor mediates its intracellular effects by coupling to defined G-protein families. Our studies have concentrated on applying a combination of molecular biological, biochemical, and cell biological approaches to define the molecular mechanisms by which adenosine receptor-derived signaling events are regulated. These studies have revealed that the desensitization of closely related adenosine receptors, which bind the same agonist in vivo (i.e., adenosine) and activate the same family of G proteins, is mediated by unique receptor-specific mechanisms. In this review, the nature of these differences, and how they could potentially be exploited to test novel strategies for enhancing the cardioprotective effects of inhibitory adenosine receptor activation in vivo, will be described. Drug Dev. Res. 58:302-314, 2003.

Research paper thumbnail of The role of sphingosine-1-phosphate in smooth muscle contraction

Cellular Signalling, 2005

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is known to mediate div... more Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is known to mediate diverse cellular responses including cell growth, survival, and migration. Most of these effects have been attributed to its binding to a specific subfamily of G protein-coupled receptors (GPCR), namely S1P(1-5). Recent studies have suggested that S1P also plays a prominent role in the contraction of various types of smooth muscle. This review provides a brief overview of its role in this process and also highlights how S1P-dependent signaling serves as an important regulator of smooth muscle contraction.

Research paper thumbnail of Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors

Blood, 2005

Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting... more Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.

Research paper thumbnail of Subtype-Specific Regulation of Receptor Internalization and Recycling by the Carboxyl-Terminal Domains of the Human A 1 and Rat A 3 Adenosine Receptors:  Consequences for Agonist-Stimulated Translocation of Arrestin3 †

Biochemistry, 2002

In this study, we have characterized the differential effects on inhibitory adenosine receptor (A... more In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys 302,305 Ala-mutated rat A 3 AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A 1 AR (Cys 309 Ala) had no effect on receptor internalization. Moreover, unlike the WT A 3 AR, the entire pool of internalized mutant A 3 AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A 3 ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A 1 AR with the 14 GRK-phosphorylated amino acids of the rat A 3 AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A 1 CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A 1 AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys 302,305 Ala-mutated A 3 ARs, as well as the A 1 CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A 1 AR nor the rat A 3 AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory ARmediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A 3 AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.

Research paper thumbnail of The S1P2 Receptor Negatively Regulates Platelet-Derived Growth Factor-Induced Motility and Proliferation

Molecular and Cellular Biology, May 15, 2005

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specif... more Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G proteincoupled receptors, named S1P 1 to S1P 5 . In this study, we found that cross-communication between plateletderived growth factor receptor and S1P 2 serves as a negative damper of PDGF functions. Deletion of the S1P 2 receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P 1 and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P 2 deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P 2 -null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P 2 reduced DNA synthesis and expression of SphK1. Thus, S1P 2 serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.