Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.
The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.
Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is speci¢city, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase CK K. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.