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Cell Biology International (2003) 27, 147–151 (Printed in Great Britain)
Regulatory role of E-NTPase/NTPDase in fat/CD36-mediated fatty acid uptake
Subburaj Kannan*
Division of Gastroenterology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA


Abstract

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


1 Introduction

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(5–50nM). Therefore, the efficient utilization of FA depends on two distinct factors; efficient transport of free FA and continuous availability without loss by either leakage or efflux from the cell to the extracellular milieu (Hajri and Abumrad, 2002).

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 40kDa plasma membrane FA-binding protein, (b) FAT/CD36, an 88kDa FA translocase, and (c) FATP, a 60kDa FA transport protein. Cytoplasmic heart-type FA-binding protein (H-FABP) has been shown to act as an intracellular carrier for FA, which probably transports FA from the sarcolemma to intracellular sites of metabolism. It remains to be determined whether free FA, binding to CD36, is deliveredby the protein directly to FA-acyl-CoA synthase inside the membrane or is transferred to another membrane protein that functions as an FA carrier. Proteins with a membrane configuration similar to CD36 have recently been shown to function in the uptake of amino acids and potassium (Hajri and Abumrad, 2002).

4 FAT/CD36

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. 14–15kDa, FABPs of cytoplasmic origin has been identified as having varied binding properties and tissue-specific expression. Based on several independent studies, it was suggested that various FABPs act together to solubilize, compartmentalize, and facilitate the cellular uptake and intracellular trafficking of FA. They act in a tissue-specific pattern, which has been shown to play a role in the modulation of mitosis, cell growth, and cell differentiation. Increased translocation of FABPpm and FAT/CD36 from intracellular stores to the membrane is thought to be the essential regulatory mechanism for increased LCFA transport, which in turn determines oxidative capacity and allows increased cardiac and skeletal muscle contraction (Luiken et al., 2000).

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 Section 5, an effort is made to elucidate an hypothetical framework to explain free FA transport via FAT/CD36.

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 90kDa). One subunit may be associated with the catalytic component of E-NTPase and the other may be of structural significance. Interaction of both the subunits or the catalytic component with FAT/CD36 may be a contributory factor in free FA transport in the myocardium.;comptd;;center;stack;;;;;6;;;;;width>

As far as the energy source is concerned, hepatic E-NTPDase (CD39), together with E-5′nucleotidase, is implicated in the regulation of [Nucleoside]0in the extracellular fluid, with a potential functional role inthe transport of metabolites between hepatic and extra-hepatic tissues. Furthermore, tetrameric CD39 complex formation is correlated with increased extracellularnucleotide triphosphate ((NTP)o) and extracellularnucleotide diiphosphate ((NDP)o) hydrolysis, and isalso thought to act as a channel for the release of NTP. These observations are termed ‘Guidotti's postulate’ (Abraham et al., 2001). It has been suggested that the rate of ATP transport from the intracellular to the extracellular milieu depends on the stoichiometry of membrane proteins, including members of the ATP-binding cassette family and CD39. CD39 could also bea pivotal molecule in providing energy (ATP releaseto the extracellular milieu, which serves as a phosphate source for ectokinase (E-kinase)/cAMP-dependent kinase), as well as an effective hydrolytic mechanism of excess (ATP)oin the circulation, to reduce E-kinase activity as the required FAT/CD36 phosphorylation is achieved.

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>


Fig. 1

Schematic view of the role of CD39 in FAT/CD36 activation.



Fig. 2

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)ois degraded by catalytically active membrane-bound and soluble CD39 and/or other uncharacterized ectonucleotidases. Other post-translational modifications may well occur in order to activate FAT/CD36. Upon forming the active complex, free FA is transported across the membrane. When FA reaches saturation level inside the cell, subsequent signaling probably terminates its transport.

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).

Acknowledgements

This work was supported by a post-doctoral fellowship from NIH Grants RO1 DK 52216 and ROI DK44237.

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Received 6 November 2001/21 August 2002; accepted 14 November 2002

doi:10.1016/S1065-6995(02)00294-9


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)