|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2003) 27, 147151 (Printed in Great Britain)
Regulatory role of E-NTPase/NTPDase in fat/CD36-mediated fatty acid uptake
Division of Gastroenterology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Fatty acid translocase (FAT)/CD36-mediated long-chain fatty acid uptake in human umbilical vessel endothelial cells is associated with as yet uncharacterized translocase activity. The molecular mechanism of its function is not yet understood. Numerous attempts to purify rat cardiac sarcolemmal E-NTPase (an integral membrane protein also referred to as ecto-Ca2+/Mg2+ATPase) have revealed a complete amino acid sequence identity for FAT/CD36 protein. The most striking observation is that purified CD36 from human platelets shows significant E-NTPase activity. In view of recent progress in understanding CD36 functional properties, an attempt is made in this article to illustrate the point that association of E-NTPase (possibly extracellular Ca2+/Mg2+nucleotide triphosphate diphosphohydrolase) activity with CD36 may be of potential functional significance.
Keywords: Fatty acid translocase/CD36, Extracellular Ca2+/Mg2+nucleotide triphosphatase, Extracellular Ca2+/Mg2+nucleotide triphosphate diphosphohydrolase, Fatty acid uptake.
*Present address: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA. Tel./fax: +1-409-750-9060
The observation that a preparation of purified CD36 protein from human platelets showed significant extracellular Ca2+/Mg2+nucleotide triphosphatase (E-NTPase) activity raised the possibility that E-NTPase and/or extracellular Ca2+/Mg2+nucleotide triphosphate diphosphohydrolase (E-NTPDase) activity may play a role in fatty acid (FA) transport mediated via FA translocase (FAT)/CD36. The amino acid sequence of plasma membrane E-NTPase (purified from rat cardiac sarcolemma light membrane) falls short of consensus (either nucleotide or amino acid) sequence similarity with E-NTPase candidate sequences, such as potato apyrase/ecto-Apyrase CD39 (Handa and Guidotti, 1996), or chicken gizzard ecto-ATPase (Kirley, 1997). Available information on CD39, in particular, points towards the hypothetical framework of a channel on its own (Abraham et al., 2001).
2 Critical appraisal of anecdotal observations
Association of E-NTPase activity with purified CD36 may be due to co-purification. It may also be due to the fact that E-NTPase activity is required for the structural stability of CD36 as a co-factor, and also for an unknown functional property. This property could be transitory in its association, depending on the structure of CD36. It is possible that CD36 acquires E-NTPase/E-NTPDase activity and, once this is achieved, E-Type nucleotidase activity is lost by dissociation from CD36 due to proteolytic degradation. To date, there is no direct experimental evidence to substantiate these conjectures. A schematic review of FA transport in conjunction with evidence pertaining to the E-NTPase/CD36 interaction is presented in subsequent discussion.
3 FA transport
β-Oxidation of FA yields metabolic energy for many cellular processes. FA uptake has been shown to bear the characteristic features (specificity, storability, and sensitivity to protein modifiers) of a carrier-mediated process, in comparison to a flip-flop (flippase) or simple passive process. Obviously, cells that are actively utilizing free FA are likely to possess a transport systemthat effectively and efficiently mediates FA uptake and targets or channels it to intracellular sites where it is required. Furthermore, it is known that unbound free FA (unFA) is present in the extracellular milieu at much lower concentrations than the albumin-bound form(5–50
At least three membrane-bound FA-binding proteins have been identified, and their functional properties pertaining to their role in FA uptake are characterized in detail: (a) FABPpm, a 40
CD36 is a membrane protein with a glycosylated extracellular domain, one (or possibly two) membrane spanning segments, and a very short intracellular segment. An adipocyte membrane glycoprotein (FAT), homologous to human CD36, has been shown to play a pivotal role in the binding/transport of long-chain FA (LCFA). FA uptake in FAT-expressing P21 cells has been shown to possess two components, namely a phloretin-sensitive high-affinity saturable component, and a basal phloretin-insensitive component that follows a linear pattern as a function of extracellular unFA concentration. Increased expression of FAT in P21 cells results in the incorporation of more exogenous FA into phospholipids, demonstrating that transfer is followed by the binding of FA to the cell membrane, andthat both processes are dependent on FAT expression. Furthermore, the data support the idea that FAT/CD36 functions as a high-affinity membrane receptor/transporter for LCFA.
Although the existence of a diffusion-like system for FA uptake is widely accepted, heterologous expression of CD36 in fibroblasts has been shown to increase LCFA uptake, despite the low concentration of free FA in the circulation. CD36 has been shown to bind to free FA with high affinity. FA-binding has also been shown to be reversible and independent of post-translational modification (palmitoylation). Based on homology between the FABPs, the amino acid residues 127–279 in the extracellular domain of CD36 appear to constitute a potential site for free FA-binding, providing efficient transport into cells that are actively utilizing FA for metabolic energy (Baillie et al., 1996; Ibrahimi and Abumrad, 2002).
Cell lines transfected with 88-kDa putative FAT (homologous to CD36) or 63-kDa FA-transport protein show an increased rate of FA uptake. Furthermore, a family of small, i.e. 14–15
It can be established, therefore, that FAT/CD36 plays a significant role in free FA transport. However, the precise molecular mechanism of this transport is not yet fully understood. In
5 Rationale for the proposed hypothesis
The transport process consists of two components; structural and functional. The structural component provides the physiologically favorable protein conformation to perform its function of sustaining homeostasis, whereas the functional component provides the appropriate signals or energy to allow optimal performance. Since FAT/CD36-mediated free FA transportdepends on the availability of free FA, which is at alow concentration in the extracellular region, obviously FAT/CD36 requires energy or post-translational modification to function as a transporter. As identified in earlier studies, cardiac E-NTPase is made up of two distinct peptides (∼80 and 90
As far as the energy source is concerned, hepatic E-NTPDase (CD39), together with E-5′nucleotidase, is implicated in the regulation of [Nucleoside]
Thus, CD39 (E-NTPase/E-NTPDase) provides structural and functional support for FAT/CD36. Indirect and circumstantial experimental evidence is provided subsequently to validate this conjecture.;comptd;;center;stack;;;;;6;;;;;width>
Schematic view of the role of CD39 in FAT/CD36 activation.
Schematic view of the role of E-kinase and CD39 in FAT/CD36 activation.
6 Indirect experimental evidence
Endothelial cells supplemented with saturated ormonounsaturated (oleic acid) FA(s) showed a marked increase in E-NTPDase activity, whereas polyunsaturated FA had an inhibitory effect. Further, it was suggested that endothelial E-NTPDase activity may be regulated by exogenous FA, and the underlying mechanism may operate via alterations in the phospholipid composition of endothelial cell membranes that control responses to oxidative stress.
7 Hypothesis: regulatory role of E-NTPase/NTPDase in FAT/CD36-mediated FA uptake
The central theme of this proposal is that the ectonucleotidases are part of the FAT/CD36 activation mechanism at the plasma membrane level. As shown in Fig. 1, the monomeric form of FAT/CD36 is translocated to the membrane surface in response to extracellular stimuli. It remains functionally inactive due to the hydrolysis of extracellular nucleotides by CD39 or other, as yet uncharacterized, cell-surface ectonucleotidases. Since FAT/CD36 is not activated to form a functional complex, obviously there is no free FA transport across the plasma membrane. If it were functional, a wide array of negative signaling cascades would have had to occur to sustain the activation of CD39 and/or other uncharacterized cell surface ectonucleotidases (shown as question marks in Fig. 1).
In comparison, in cells that are actively utilizing free FA as a source of energy, the scheme that is illustrated in Fig. 2 is probably functional. Briefly, in response to extracellular stimuli, the monomeric form of FAT/CD36 is translocated to the membrane surface. It is subsequently phosphorylated by E-kinase/cAMP-dependent kinase, resulting in oligomerization or functionally active FAT/CD36. The remaining excess (ATP)
CD39 is the most likely candidate to be involved in this regulation because, as Guidotti's postulate projected, it could act as both ATP release and hydrolysis systems with higher efficiency than any other regulatory mechanism. If this were to result in a functional complex, a wide array of positive-feedback signaling cascades would need to occur in order to sustain the activation of E-kinase/cAMP-dependent kinase and CD39 (or other uncharacterized cell surface ectonucleotidases).
Bile acid efflux and Ca2+/Mg2+ecto-ATPase activities have been shown to be two distinct properties of a single rat liver hepatocyte canalicular membrane protein. However, introduction of mutations in the consensus sequence in amino acids Gly 97 and Arg 98 in the Ca2+/Mg2+ecto-ATPase abrogated ATPase activity, but did not affect bile acid transport activity (Sippel et al., 1994a,b). Electrogenic taurocholate transport has been shown to be an intrinsic function of the canalicular membrane, together with an as yet unidentified intracellular membrane-bound compartment. Therefore, the two transport activities are probably mediated by two different bile acid transporting polypeptides. In the context of the present proposal, FAT/CD36 may be the free FA transport system, while CD39 is the energy-providing source (Kast et al., 1994).
This work was supported by a post-doctoral fellowship from NIH Grants RO1 DK 52216 and ROI DK44237.
Abraham EH, Sterling, KM, Kim, AY, Salikhova, HB, Huffman, MA, Crockett, N. Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis. Blood Cells Mol Dis 2001:27:165-80
Kast C, Stieger, B, Winterhalter, KH, Meier, PJ. Hepatocellular transport of bile acids. Evidence for distinct subcellular localizations of electrogenic and ATP-dependent taurocholate transport in rat hepatocytes. J Biol Chem 1994:269:5179-86
Kirley TL. Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. Homology with the lymphoid cell activation antigen CD39. J Biol Chem 1997:272:1076-81
Luiken JJ, Glatz, JF, Bonen, A. Fatty acid transport proteins facilitate fatty acid uptake in skeletal muscle. Can J Appl Physiol 2000:25:333-52
Sippel CJ, Fallon, RJ, Perlmutter, DH. Bile acid efflux mediated by the rat liver canalicular bile acid transport/ecto-ATPase protein requires serine 503 phosphorylation and is regulated by tyrosine 488 phosphorylation. J Biol Chem 1994:269:19539-45
Sippel CJ, McCollum, MJ, Perlmutter, DH. Bile acid transport by the rat liver canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ecto-ATPase activity. J Biol Chem 1994:269:2820-6
Received 6 November 2001/21 August 2002; accepted 14 November 2002doi:10.1016/S1065-6995(02)00294-9