Active 3ʹ–5ʹ cyclic nucleotide phosphodiesterases are present in detergent-resistant membranes of mural granulosa cells

Lipids rafts are specialised membrane microdomains involved in cell signalling that can be isolated as detergent-resistant membranes (DRMs). The second messenger cyclic AMP (cAMP) has a central role in cell signalling in the ovary and its degradation is carried out by the phosphodiesterase (PDE) enzyme family. We hypothesised that PDEs could be functionally present in the lipid rafts of porcine mural granulosa cell membranes. PDE6C, PDE8A and PDE11A were detected by dot blot in the DRMs and the Triton-soluble fraction of the mural granulosa cells membrane and the cytosol. As shown by immunocytochemistry, PDEs showed clear immunostaining in mural granulosa cell membranes and the cytosol. Interestingly, cAMP–PDE activity was 18 times higher in the DRMs than in the Triton-soluble fraction of cell membranes and was 7.7 times higher in the cytosol than in the DRMs. cAMP–PDE activity in mural granulosa cells was mainly contributed by the PDE8 and PDE11 families. This study shows that PDEs from the PDE8 and PDE11 families are present in mural granulosa cells and that the cAMP–PDE activity is mainly contributed by the cytosol. In the cell membrane, the cAMP–PDE activity is mainly contributed by the DRMs. In addition, receptors for prostaglandin E2 and LH, two G-protein-coupled receptors, are present in lipid rafts and absent from the non-raft fraction of the granulosa cell membrane. These results suggest that in these cells, the lipid rafts exist as a cell-signalling platform and PDEs are one of the key enzyme families present in the raft.

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Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Circulation ◽

10.1161/circ.116.suppl_16.ii_83-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

David R Graham ◽

Antoine Younes ◽

Alexey Lyashkov ◽

Anna Sheydina ◽

Maria Volkova ◽

...

Keyword(s):

Lipid Rafts ◽

Sucrose Gradient ◽

Soluble Fraction ◽

Membrane Microdomains ◽

Membrane Domains ◽

Lipid Domains ◽

Rt Pcr ◽

Pacemaker Cells ◽

Pacemaker Function ◽

Sucrose Gradient Ultracentrifugation

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.

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Cell Membrane Sulfatide Promotes Sickle Cell Adhesion to Endothelium.

Blood ◽

10.1182/blood.v110.11.1722.1722 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1722-1722

Author(s):

Prasenjit Guchhait ◽

Perumal Thiagarajan ◽

Jose A. Lopez

Keyword(s):

Cell Adhesion ◽

Cell Membrane ◽

Lipid Rafts ◽

Sickle Cell ◽

Membrane Microdomains ◽

Shear Stresses ◽

Single Chain ◽

Intense Staining ◽

Sickle Cells ◽

Initial Adhesion

Abstract Sickle cell disease (SCD) affects millions of people worldwide, and is associated with significant morbidity and mortality. Although the clinical manifestations of the disease are very complex, much of the cause can be ascribed to occlusion of small vessels by the sickle red blood cells (RBCs). More than 30% of all deaths in SCD are due to the vasoocclusion, which results in ischemia, multiorgan failure and strokes. The proximate cause for vasoocclusion appears to be an increased adhesiveness of sickle cells to the vessel wall, and we postulate that the exposure of sulfatide on sickle cells accounts for their adhesive phenotype. Sulfatide binds with high affinity to many of the adhesion proteins known to be involved in cell adhesion to subendothelium and endothelium, including von Willebrand factor (VWF), thrombospondin (TSP), laminin and P-selectin. We therefore compared the expression and distribution of sulfatide in sickle cells to that in normal RBCs. When examined by flow cytometry using a previously described Alexa fluor-conjugated single-chain variable fragment (scFv) antibody, PA38, we found that sickle cells displayed more sulfatide on surface than normal RBCs (mean fluorescence 1.6±0.5 Vs. 0.9±0.3, p<0.05, n=6). When we examined sulfatide distribution by confocal microscopy using the labeled PA38, we found it to label more intensely in sickle cells than the normal RBCs and to be distributed heterogeneously, with areas of intense staining. The heterogeneous distribution suggested that the sulfatide might exist within membrane-microdomains/lipid rafts. We tested this possibility by sucrose density centrifugation of detergent lysates (1% Triton X-100) of erythrocyte ghosts from sickle and normal cells and found that sulfatide was distributed in raft fractions, as defined by being in the fractions containing the raft marker flotillin-1. Consistent with an important role for sulfatide in sickle vaso-occlusion, we found that both normal and sickle RBCs attached under flow to the surface of histamine-activated human umbilical vein endothelial cells (HUVEC). The sickle RBCs adhered more avidly, as they were able to rest higher shear stresses (1.86 and 2.5 dyne/cm2) than the normal RBCs before detaching Greater than 50% of the initial adhesion was inhibited by treatment with the anti-sulfatide scFv, PA38. We obtained similar results in terms of the greater shear resistance of sickle cells and the ability of PA38 to inhibit adhesion when we compared the adhesion of sickle and normal RBCs to surfaces coated with the adhesive ligands such as VWF (the ultra-large form) and laminin. Thus, our study elucidates an important role of red cell membrane sulfatide in sickle cell adhesion to the endothelium and to adhesive ligands, and suggests that this mechanism is important pathophysiologically in the development of sickle vaso-occlusion. Sulfatide distribution into lipid rafts may allow the formation of adhesive patches that facilitate adhesion.

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Changes in receptor location affect the ability of oxytocin to stimulate proliferative growth in prostate epithelial cells

Reproduction Fertility and Development ◽

10.1071/rd18362 ◽

2019 ◽

Vol 31 (6) ◽

pp. 1166 ◽

Cited By ~ 2

Author(s):

M. L. Gould ◽

H. D. Nicholson

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Cell Membrane ◽

Lipid Rafts ◽

Protein Interactions ◽

Cell Signalling ◽

Signalling Pathways ◽

Protein Protein Interactions ◽

Normal Prostate ◽

Prostate Epithelial Cells

In normal prostate cells, cell membrane receptors are located within signalling microdomains called caveolae. During cancer progression, caveolae are lost and sequestered receptors move out onto lipid rafts. The aim of this study was to investigate whether a change in the localisation of receptors out of caveolae and onto the cell membrane increased cell proliferation invitro, and to determine whether this is related to changes in the cell signalling pathways. Normal human prostate epithelial cells (PrEC) and androgen-independent (PC3) cancer cells were cultured with 10nM dihydrotestosterone (DHT). The effects of oxytocin (OT) and gonadal steroids on proliferation were assessed using the MTS assay. Androgen receptor (AR) and oxytocin receptor (OTR) expression was identified by immunofluorescence and quantified by western blot. OTR and lipid raft staining was determined using Pearson’s correlation coefficient. Protein–protein interactions were detected and the cell signalling pathways identified. Treatment with OT did not affect the proliferation of PrEC. In PC3 cells, OT or androgen alone increased cell proliferation, but together had no effect. In normal cells, OTR localised to the membrane and AR localised to the nucleus, whereas in malignant cells both OTR and AR were identified in the cell membrane. Colocalisation of OTR and AR increased following treatment with androgens. Significantly fewer OTR/AR protein–protein interactions were seen in PrEC. With OT treatment, several cell signalling pathways were activated. Movement of OTR out of caveolae onto lipid rafts is accompanied by activation of alternative signal transduction pathways involved in stimulating increased cell proliferation.

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Lipid Rafts: Linking Alzheimer's Amyloid-βProduction, Aggregation, and Toxicity at Neuronal Membranes

International Journal of Alzheimer s Disease ◽

10.4061/2011/603052 ◽

2011 ◽

Vol 2011 ◽

pp. 1-14 ◽

Cited By ~ 34

Author(s):

Jo V. Rushworth ◽

Nigel M. Hooper

Keyword(s):

Glutamate Receptors ◽

Lipid Rafts ◽

Lipid Raft ◽

Cell Signalling ◽

Cellular Prion Protein ◽

Membrane Microdomains ◽

Synaptic Dysfunction ◽

Memory Impairments ◽

Neuronal Membranes ◽

Neuronal Functions

Lipid rafts are membrane microdomains, enriched in cholesterol and sphingolipids, into which specific subsets of proteins and lipids partition, creating cell-signalling platforms that are vital for neuronal functions. Lipid rafts play at least three crucial roles in Alzheimer's Disease (AD), namely, in promoting the generation of the amyloid-β(Aβ) peptide, facilitating its aggregation upon neuronal membranes to form toxic oligomers and hosting specific neuronal receptors through which the AD-related neurotoxicity and memory impairments of the Aβoligomers are transduced. Recent evidence suggests that Aβoligomers may exert their deleterious effects through binding to, and causing the aberrant clustering of, lipid raft proteins including the cellular prion protein and glutamate receptors. The formation of these pathogenic lipid raft-based platforms may be critical for the toxic signalling mechanisms that underlie synaptic dysfunction and neuropathology in AD.

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Hormone-induced intercellular signal transfer dissociates cyclic AMP-dependent protein kinase.

The Journal of Cell Biology ◽

10.1083/jcb.98.5.1710 ◽

1984 ◽

Vol 98 (5) ◽

pp. 1710-1719 ◽

Cited By ~ 67

Author(s):

S A Murray ◽

W H Fletcher

Keyword(s):

Protein Kinase ◽

Cell Membrane ◽

Gap Junctions ◽

Granulosa Cell ◽

Cell Fusion ◽

Granulosa Cells ◽

Signal Transfer ◽

Dependent Protein Kinase ◽

Ovarian Granulosa Cells ◽

Dependent Protein

We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact-dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and occurs in the absence of apparent cell fusion. The only structural feature connecting Y-1 and granulosa cells were gap junctions implying they provided the communication channels; however, alternative mechanisms cannot be excluded. We have not established the identity of the signal being transferred although cAMP is a logical candidate.

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Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosisThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Biochemistry and Cell Biology ◽

10.1139/o06-180 ◽

2006 ◽

Vol 84 (6) ◽

pp. 993-1005 ◽

Cited By ~ 22

Author(s):

Lillian S. DeBruin ◽

Jeffery D. Haines ◽

Dorothee Bienzle ◽

George Harauz

Keyword(s):

Myelin Basic Protein ◽

Lipid Rafts ◽

Basic Protein ◽

Membrane Microdomains ◽

Special Issue ◽

Splice Isoforms ◽

Severity Of Disease ◽

Health And Disease ◽

Detergent Resistant Membranes ◽

Selection Of

We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14–21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.

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Mechanisms underlying the immunomodulatory effects of n-3 PUFA

Proceedings of The Nutrition Society ◽

10.1017/s0029665110001837 ◽

2010 ◽

Vol 69 (3) ◽

pp. 311-315 ◽

Cited By ~ 17

Author(s):

Parveen Yaqoob

Keyword(s):

Immune Function ◽

Lipid Rafts ◽

Cell Membranes ◽

Immune Cell ◽

Human Lymphocytes ◽

Membrane Microdomains ◽

Future Research ◽

Immunomodulatory Effects ◽

The Impact

The enrichment of immune cell membranes with n-3 PUFA is associated with modulation of immune function. The degree of incorporation of n-3 PUFA (and therefore the impact of dietary n-3 PUFA on immune function) appears to depend on a number of factors including species and age. The mechanisms involved are still largely unclear, but recent work has focused on two areas; lipid rafts and eicosanoids. In vitro studies suggest that lipid rafts could play a role in the immunomodulatory effects of n-3 PUFA, but there is still little information regarding the extent to which membrane microdomains in human lymphocytes are modulated by dietary supplementation. The enrichment of cell membranes with n-3 PUFA also modulates the production of eicosanoids, the full extent of which has not yet been realized; this represents a key area for future research.

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Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

Essays in Biochemistry ◽

10.1042/bse0570189 ◽

2015 ◽

Vol 57 ◽

pp. 189-201 ◽

Cited By ~ 13

Author(s):

Jay Shankar ◽

Cecile Boscher ◽

Ivan R. Nabi

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Cancer Cell ◽

Cellular Response ◽

Spatial Organization ◽

Essential Feature ◽

Cell Signalling ◽

Coated Pits ◽

Signalling Molecules ◽

Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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