Download The monocarboxylate transporter family

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Life, 64(1): 1–9, January 2012
Critical Review
The Monocarboxylate Transporter Family—Structure and Functional
Characterization
Andrew P. Halestrap
School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol, UK
Abbreviations
Summary
Monocarboxylate transporters (MCTs) catalyze the protonlinked transport of monocarboxylates such as L-lactate, pyruvate, and the ketone bodies across the plasma membrane. There
are four isoforms, MCTs 1–4, which are known to perform this
function in mammals, each with distinct substrate and inhibitor
affinities. They are part of the larger SLC16 family of solute
carriers, also known as the MCT family, which has 14 members
in total, all sharing conserved sequence motifs. The family
includes a high-affinity thyroid hormone transporter (MCT8),
an aromatic amino acid transporter (T-type amino acid transporter 1/MCT10), and eight orphan members yet to be characterized. MCTs were predicted to have 12 transmembrane helices (TMs) with intracellular C- and N-termini and a large intracellular loop between TMs 6 and 7, and this was confirmed
by labeling studies and proteolytic digestion. Site-directed mutagenesis has identified key residues required for catalysis and inhibitor binding and enabled the development of a molecular
model of MCT1 in both inward and outward facing conformations. This suggests a likely mechanism for the translocation
cycle. Although MCT family members are not themselves glycosylated, MCTs1–4 require association with a glycosylated ancillary protein, either basigin or embigin, for their correct translocation to the plasma membrane. These ancillary proteins have a
single transmembrane domain and two to three extracellular
immunoglobulin domains. They must remain closely associated
with MCTs1–4 to maintain transporter activity. MCT1, MCT3,
and MCT4 bind preferentially to basigin and MCT2 to embigin.
The choice of binding partner does not affect substrate specificity or kinetics but can influence inhibitor specificity. Ó 2011
IUBMB
IUBMB
Keywords
Life, 64(1): 1–9, 2012
lactate; pyruvate; metabolism; MCT1; MCT2; MCT3;
MCT4; MCT8; thyroid hormone; basigin; embigin.
Received 9 May 2011; accepted 8 August 2011
Address correspondence to: Andrew P. Halestrap, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol
BS8 1TD, United Kingdom. E-mail: [email protected]
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.573
BCECF, 20 -70 -bis(carboxyethyl)-5-6-carboxy-fluorescein; CHC, a-cyano-4-hydroxycinnamate; DBDS,
4,40 -dibenzamidostilbene-2,20 -disulfonate; DIDS, 4,40 di-isothiocyanostilbene-2,20 -disulfonate; MCT, monocarboxylate transporter; pCMBS, p-chloromercuribenzenesulfonate; TM, transmembrane helix.
INTRODUCTION
Monocarboxylates such as pyruvate, lactate, and the ketone
bodies (acetoacetate and b-hydroxybutyrate) play essential roles
in carbohydrate, fat, and amino acid metabolism and must be
rapidly transported across the plasma membrane of cells (1, 2).
Transport is mediated by proton-linked monocarboxylate transporters (MCTs), four of which (MCTs1–4) have been characterized in detail and will be the major focus of this review. They
are part of a family of transporter proteins, known as the MCT
or SLC16 solute carrier family, that share characteristic
sequence motifs. All family members are predicted to have 12transmembrane helices (TMs) with intracellular C- and N-termini and a large cytosolic loop between TMs 6 and 7. As in
other major facilitator superfamily members, the TM regions
are more conserved than the loops and C-terminus [see (2–4)].
The MCT family has a total of 14 members whose predicted
phylogeny is shown in Fig. 1 but only MCTs1–4 have been
confirmed to function as proton-linked MCTs. MCT10 was
identified as an aromatic amino acid transporter originally called
T-type amino acid transporter 1 (TAT1) (5) and MCT8 shown
to be an important thyroid hormone transporter (6). Transport
mediated by MCT8 and MCT10 is not proton linked. The function of the other eight MCTs is unknown (2, 4). In addition to
their normal metabolic roles, which are summarized in Fig. 2,
MCTs may also be important for the transport of some drugs
across the plasma membrane (4). There are also two distinct sodium-linked monocarboxylate transporters that are members of
the SLC5 solute carrier family. These play a key role in endothelial monocarboxylate transport in the gut and kidney and will
2
HALESTRAP
Figure 1. The predicted phylogeny of MCT family members.
For each family member, both the MCT and SLC16 numbers
are given. Six of the 14 members have been functionally characterized with only MCT1–4 showing proton-coupled lactate
transport, whereas MCT8 and MCT 10 catalyze the sodiumand proton-independent transport of thyroid hormone and aromatic acids, respectively. The properties of the other eight
members remain to be elucidated but we have failed to demonstrate proton-linked transport of a wide range of monocarboxylates by MCT5 or MCT7 expressed in oocytes. The figure is a
modified version of that presented in (2). The bar indicates the
number of substitutions per residue with 0.1 corresponding to a
distance of 10 substitutions per 100 residues.
not be discussed here but have been well reviewed elsewhere
(7).
This article will briefly review what is known of the structure
and properties of the well-characterized members of the MCT
family: MCT1 (SLC16A1), MCT2 (SLC16A7), MCT3
(SLC16A8), MCT4 (SLC16A3), MCT8 (SLC16A2), and MCT10
(SLC16A10). A subsequent article (8) will review their tissue
specific metabolic roles and regulation. At the outset, the reader
should note that the nomenclature of the MCT family is confusing, as in many cases, the MCT and SLC16 numbering do not
coincide. This discrepancy arose because the MCTs were named
in order of their characterization at the functional level, while the
SLC16 numbers were generated as the cDNA sequences became
available. The reader is referred to a previous review (3) for
additional information on how these nomenclature issues arose
fuller account of the current nomenclature of all members of the
SLC16/MCT family with details of the chromosomal location
and gene structure of the human MCTs is in (2). The accompanying article will review the metabolic roles and regulation of
members of the MCT family (8).
Figure 2. The role of MCTs in metabolism. It should be noted
that no single cell will carry out the full spectrum of pathways
shown. Depending on the tissue and the species, MCT1 or
MCT2 are used to take up lactic acid and ketone bodies for oxidation (e.g., heart, red muscle, and neurons) or lactic acid for
gluconeogenesis (liver and kidney). In most tissues that rely on
glycolysis for their energy metabolism under normoxic conditions (e.g., white skeletal muscle fibers), lactic acid efflux utilizes MCT4, but MCT3 fulfils this function in the retinal pigment epithelium. All cells export lactic acid under hypoxic conditions and use whichever MCT isoform is expressed (normally
MCT1). Further details are presented in the text and refs. 1–4.
A more detailed consideration of tissue specific roles of MCTs
is giving in the accompanying review where the role of different MCT isoforms in shuttling lactate between different cell
types within a tissue is discussed (8).
CHARACTERIZATION OF THE DIFFERENT
MCT FAMILY MEMBERS
MCT1
The existence of a proton-linked monocarboxylate transporter was first demonstrated in this laboratory by showing that
transport of L-lactate and pyruvate into human red blood cells
was specifically inhibited by a-cyano-4-hydroxycinnamate
(CHC) (9). The substrate and inhibitor specificity and detailed
kinetics of this transporter were extensively characterized in
both the author’s and Deuticke’s laboratories [see (1, 10)]. Subsequently, the molecular identity of the protein responsible was
established by protein purification and sequencing in this laboratory (11) and independently at the cDNA level by Christine
Kim Garcia in the laboratory of Goldstein and Brown. These
authors named the transporter MCT1 (12). MCT1 is found in
the great majority of tissues in all species studied, with no evidence for splice variants within the coding region (2, 4).
Substrate Specificity. Characterization of the properties of
MCT1 was initially performed in human and rat erythrocytes
using radioactive techniques [see (1, 10)] and confirmed in a
MCT STRUCTURE AND FUNCTION
3
Table 1
Km Values of different MCT isoforms for a range of monocarboxylates
Substrate
MCT1 tumor cells
MCT1 oocytes
MCT2 oocytes
MCT3 yeast
MCT4 oocytes
Formate
Bicarbonate
Oxamate
Glyoxylate
L-Lactate
D-Lactate
Pyruvate
S-Chloropropionate
R-Chloropropionate
D,L-a-Hydroxybutyrate
L-b-Hydroxybutyrate
D-b-Hydroxybutyrate
c-Hydroxybutyrate
Acetoacetate
a-Ketobutyrate
a-Ketoisocaproate
a-Ketoisovalerate
b-Phenylpyruvate
[100
–
49
63
4.5
27.5
0.7
0.7
0.7
2.6
11.4
10.1
7.7
5.5
0.2
–
–
–
–
–
–
–
3.5
[60
1.0
–
–
–
–
–
–
–
–
0.7
1.3
–
–
–
–
–
0.74
–
0.08
–
–
–
1.2a
1.2a
–
0.8
–
0.1
0.3
–
–
–
–
–
6
–
–
–
–
–
–
–
–
–
–
–
–
–
[500b
[500b
[500b
[500b
28
519
153
46
51
56
824
130
[500b
216
57
95
113
[500b
Data are for Km values (mM) of endogenous MCT1 in tumor cells or for MCT1, MCT2, and MCT4 expressed in Xenopus oocytes as indicated and are
taken from ref. 14 where further details may be found. The L-lactate Km for MCT3 was measured following expression in Yeast and taken from ref. 16.
a
D,L-Racemic mix used in these studies.
b
Uptake at 50 mM is very low to measure.
mouse tumor cell line by monitoring changes in intracellular pH
using the fluorescent pH indicator 20 -70 -bis-(carboxyethyl)-5-6carboxy-fluorescein (BCECF) (13). Subsequently, MCT1 was
expressed in Xenopus laevis oocytes that exhibit no significant
endogenous MCT activity and its activity again determined either radioactively or by monitoring transport-mediated changes
in intracellular pH with BCECF or pH microelectrodes (14, 15).
All these techniques confirmed that MCT1 demonstrates
Michaelis Menten kinetics with a broad specificity for shortchain monocarboxylates including those substituted on the 2
and 3 positions with small groups such as halides, hydroxyl,
and carbonyl groups as illustrated in Table 1. In addition, the
transport of unsubstituted short-chain fatty acids, such as acetate, propionate, and butyrate, is strongly facilitated by MCT1,
but these substrates can also enter cells rapidly by free diffusion
of the undissociated acid [see (1, 10)]. Natural occurring substrates for MCT1 include L-lactate, pyruvate, b-hydroxybutyrate, and acetoacetate (1, 2) and Km values for these substrates
are within the range found physiologically (1, 8). More hydrophobic ketoacids derived by transamination of amino acids may
also be transported by MCT1; these include phenylpyruvate
(from phenylalanine), a-ketoisocaproate (from leucine), a-ketoisovalerate (from valine), and a-keto-b-methylvalerate (from isoleucine). However, the hydrophic side chain of these substrates
impairs the release of the bound substrate following transport
resulting in very slow rates of net transport of these substrates
by MCT1; indeed, they act as potent competitive inhibitors of
the transport of other monocarboxylates (13). Interestingly, the
transport of lactate is relatively stereoselective, with D-lactate
being a poor substrate compared to L-lactate, whereas such stereoselectivity is not demonstrated for 2-chloropropionate or
b-hydroxybutyrate. By far, the predominant role of MCT1 is to
facilitate unidirectional proton-linked transport of L-lactate
across the plasma membrane. This may represent either influx
or efflux of lactic acid depending of the prevailing intracellular
and extracellular substrate concentrations and the pH gradient
across the plasma membrane. Net rates of transport of any
monocarboxylate will be determined by the difference between
influx and efflux, and at thermodynamic equilibrium, the concentration ratio of monocarboxylate inside the cell to outside
the cell is equal to the ratio of [H1]out to [H1]in. However,
MCT1 can also exchange one monocarboxylate for another
without net movement of protons [see (1–3)].
Inhibitors of MCT1. In addition to inhibition by competing
monocarboxylates such as those described above (Substrate
Specificity section), numerous nonphysiological competitive
inhibitors of MCT1 have been described. These include CHC
analogs and stilbene disulfonates such as 4,40 -di-isothiocyanostilbene-2,20 -disulfonate (DIDS) and 4,40 -dibenzamidostilbene2,20 -disulfonate (DBDS) [see (1, 10)]. These agents have been
used in some published experiments as specific MCT1 inhibitors
4
HALESTRAP
without recognizing their ability to act as significant powerful
inhibitors of other transporters. Thus, CHC is at least two orders
of magnitude more potent as an inhibitor of the mitochondrial
pyruvate transporter than of MCT1, whereas DIDS and DBDS
inhibit the chloride/bicarbonate exchanger AE1 more powerfully
than MCT1. The noncompetitive inhibitor phloretin and the arginine and cysteine reactive reagents phenylglyoxal and p-chloromercuribenzenesulfonate (pCMBS) [see(1, 10)] are equally
nonspecific.
A new class of specific and high-affinity inhibitors of MCT1
have recently been developed by AstraZeneca with Ki values in
the nM region (17, 18). We have confirmed the potent inhibition
of MCT1-mediated L-lactate transport into rat erythrocytes by
one of these inhibitors, AR-C155858, and determined a Ki of
about 2 nM (19). This very high-affinity enabled us to determine
the number of molecules of MCT1 per erythrocyte (80,000) and
the turnover number (kcat) of the transporter (12 s21 at 68C). We
also characterized the inhibition by AR-C155858 of different
MCT isoforms expressed in Xenopus oocytes and found it to be
active against MCT1 and MCT2 but not MCT4. Comparison of
the time dependence of inhibition following extracellular application or intracellular injection of AR-C155858 suggested that it
binds to MCT1 from the cytosolic side. The use of chimeric
transporters combining different domains of MCT1 and MCT4
revealed that the binding site for the inhibitor is contained within
the C-terminal half of MCT1, and involves TM domains 7–10
(19). Subsequent studies on the inhibition of MCT2 by ARC155858 revealed that inhibition of this isoform only occurred
when MCT2 is associated with the ancillary protein basigin and
not when associated with embigin (see Some MCTs Require an
Ancillary Protein for Plasma Membrane Expression and Activity
section), whereas the choice of ancillary protein has no effect on
the inhibition of MCT1 (19).
Molecular Mechanism of MCT1. The molecular mechanism by
which MCT1 transports monocarboxylates has been extensively
studied by analysis of transport kinetics, the use of inhibitors
and more recently through site-directed mutagenesis. Detailed
kinetic analysis of monocarboxylate transport in erythrocytes
revealed that MCT1 operates through an ordered mechanism
[see (1, 10)], although a recent analysis of published data of
lactate transport into sarcolemmal vesicles and oocytes mediated
by MCT1 and MCT4 concluded that it is not possible to discriminate between an ordered and a random binding mechanism
(20). However, it is surprising that these authors did not refer to
the more rigorous erythrocyte studies described above that
strongly support an ordered mechanism. The ordered mechanism predicts that when transporting lactate into the cell, MCT1
has a substrate binding site open to the extracellular matrix
which binds a proton first followed by the lactate anion. The
protein then undergoes a conformational change to a new
‘‘closed’’ conformation that exposes both the proton and lactate
to the opposite surface of the membrane where they are
released, lactate first and then the proton. For net transport of
lactic acid, the rate-limiting step is the return of MCT1 without
bound substrate to the open conformation. For this reason,
exchange of one monocarboxylate inside the cell with another
outside is considerably faster than net transport of a monocarboxylate across the membrane (1, 10). We have recently developed a structural model for MCT1 based on site-directed mutagenesis and homology modeling that can account for this mechanism (21, 22). This is described in Structure and Translocation
Cycle of MCTs section below.
MCT2
Even before the identification of MCT1, characterization of
monocarboxylate transport into isolated rat liver cells (23) and
heart cells (24) led us to propose that there might be several
MCT isoforms. This was confirmed when Christine Kim Garcia
in the laboratory of Goldstein and Brown cloned a second isoform which transported lactate and pyruvate when expressed in
insect Sf9 cells (25). They named this isoform MCT2 and
reported that it had a higher affinity for substrates than MCT1.
This was subsequently confirmed when rat MCT2 was characterized following expression in Xenopus oocytes (26). Km values for pyruvate and L-lactate were found to be about 0.1 and
0.74 mM, respectively, compared to values of about 1 and 3.5
mM for MCT1 (see Table 1). MCT2 was also reported to be
more sensitive than MCT1 to inhibition by a range of inhibitors
including CHC, DBDS, and DIDS but insensitive to the organomercurial reagent pCMBS (25, 26). Subsequent work in this
laboratory has shown that pCMBS actually inhibits MCT1 by
binding to the ancillary protein, basigin (see Some MCTs
Require an Ancillary Protein for Plasma Membrane Expression
and Activity section) and that MCT2 is insensitive because it
usually associates with embigin rather than basigin (27).
The sequence of MCT2 is far less conserved across species
than that of MCT1 or MCT4 and there also appear to be considerable species differences in the tissue expression profile of
this isoform (2). Both Northern blot analysis and inspection of
the human expressed sequence tag (EST) database suggest relatively little expression of MCT2 in human tissues. However, in
mouse, rat, and the hamster, both Northern and Western blot
analyses and immunofluorescence microscopy show the protein
to be expressed in liver, kidney, brain, and sperm tails, whereas
in hamster, there is also evidence for its presence in skeletal
muscle and heart (3, 25, 28). Where MCT2 is expressed together with MCT1, its exact location within the tissue is different, suggesting a unique functional role related to its high substrate affinity [see (2, 4)]. This may be especially important in
the brain where MCT2 expression is largely confined to the
postsynaptic density of the neurons and may facilitate the
uptake of lactate for oxidation as a respiratory fuel (29, 30).
MCT3
MCT3 was originally identified by Philp and coworkers
(16) as a developmentally expressed protein in the chick retinal
MCT STRUCTURE AND FUNCTION
pigment epithelium but subsequently identified as a member of
the MCT family and named MCT3. Its transport activity was
confirmed by expression in yeast where it was reported to demonstrate a Km for L-lactate of about 6 mM and to be insensitive
to CHC, phloretin, and pCMBS (31). MCT3 has yet to be characterized in Xenopus oocytes and detailed knowledge of its substrate and inhibitor specificity is lacking. Expression of MCT3
is confined to the retinal pigment epithelium and choroid plexus
epithelia (32), where it is located on the basal membrane in
contrast to MCT1 which is found on the apical membrane (33,
34). It is thought to play an important role in facilitating the
transport of glycolytically derived lactic acid out of the retina.
MCT4
MCT4 was identified in this laboratory during a search of the
EST database for novel members of the MCT family (35) and
was originally called MCT3 based on its sequence homology with
chick MCT3. It was renamed MCT4 when a distinct mammalian
MCT3 was identified in mammalian retinal pigment epithelium
[see (2)]. Northern and Western blotting and EST database analyses showed MCT4 to be widely expressed and especially so in
glycolytic tissues such as white skeletal muscle fibers, astrocytes,
white blood cells, chondrocytes, and some mammalian cell lines
[see (2–4)]. In the rat, MCT4 is expressed in the neonatal heart,
which is more glycolytic in its energy metabolism than the adult
heart where MCT4 is absent but MCT1 abundant (36, 37). This
led us to propose that the properties of MCT4 might be especially
appropriate for export of lactic acid derived from glycolysis and
characterization of its properties by expression in Xenopus
oocytes suggest this to be the case (15, 38). MCT4 exhibits a
lower affinity for most substrates and inhibitors than MCT1, with
Km and Ki values some 5–10-fold higher. Thus, Km values for Llactate and pyruvate were determined to be 28 and 150 mM,
respectively (see Table 1), and little inhibition by DIDS or CHC
was observed at concentrations giving [50% inhibition of
MCT1. The high Km for pyruvate may be especially significant as
this avoids loss of pyruvate from the cell which, were it to occur,
would prevent removal of the reduced form of nicotinamide adenine dinucleotide (NADH) produced in glycolysis by reduction of
pyruvate to lactate (see ref. 2,8).
MCT8
MCT8 (SLC16A2) was first cloned in 1994 by Lafreniere
et al. and called XPCT because it contains a long N-terminus
‘‘PEST" domain, a proline/glutamate-rich region thought to
mediate rapid proteolytic degradation (39). Following the identification of the closely related protein TAT1 (MCT10) as an aromatic amino acid transporter (see MCT10 section below), collaborative studies with the laboratory of Theo Visser enabled us
to identify MCT8 as an active and specific iodothyronine (thyroid hormone) transporter. Expression of MCT8 in Xenopus
oocytes revealed that both T4 and T3 were transported with
high affinity (Km 5 2–5 lM) whilst aromatic amino acids were
5
not transported (40). Transport is Na independent, and there is
no evidence for proton dependence. MCT8 is expressed in most
tissues, including liver, kidney, heart, skeletal muscle, brain,
pituitary, and thyroid and probably occurs naturally as a homodimer [see (41)]. Its importance in thyroid hormone transport
has been confirmed by the identification of individuals with
mutations in the MCT8 gene that exhibit major psychomotor
retardation (42).
MCT10
MCT10 (SLC16A10), also known as TAT1, was discovered
by expression cloning in Xenopus oocytes and shown to transport aromatic amino acids (phenylalanine, tyrosine, and tryptophan) in a sodium and proton independent manner with Km values of about 1 mM (5). The original studies failed to show
transport of thyroid hormone by MCT10 but subsequently such
activity was demonstrated [see (41)]. The expression MCT10 is
species dependent but it is usually strongly expressed in the
intestine, kidney, liver, skeletal muscle, heart, and placenta [see
(4)].
STRUCTURE AND TRANSLOCATION CYCLE OF MCTs
Hydrophobicity plots predict that MCTs should contain 12
TMs with intracellular C- and N-termini and a large intracellular loop between TMs 7 and 8. Indeed, proteolytic cleavage and
labeling studies of MCT1 were entirely consistent with this topology (43). Sequence analysis suggests that MCTs are unlikely
to be glycosylated (43) and no experimental evidence has been
reported that would argue otherwise. Thus, bands on Western
blots are not broad and cannot be shifted by deglycosylation
(44). Using molecular modeling, we have generated a probable
three dimensional structure of MCT1 in the ‘‘closed’’ conformation (with substrate binding site exposed to cytosol) based on
the published structure of the E Coli glycerol phosphate transporter GlpT (1PW4) (21). This model, which is shown in
Fig. 3, is consistent with results of extensive site-directed mutagenesis studies of MCT1 (reviewed in (21, 22). To model
MCT1 in the ‘‘open’’ conformation (with the substrate binding
site exposed to the extracellular medium), we used site-directed
mutagenesis to identify key lysine residues involved in the binding of DIDS which inhibits MCT1 activity by competing with
the monocarboxylate for its extracellular binding site (22). In
our model, the 6-helix N-terminal domain and the similar 6-helix C-terminal domain of MCT1 are linked by a 30-residue loop
of unknown, or no, structure. Interconversion between the two
conformations is predicted to occur by a modest reorganization
of the interface between the two domains (22) as originally suggested by Bröer et al. (45). Evidence has been provided for similar domain rearrangements in the E Coli transporter Lac-Permease during its catalytic cycle [see (46)].
The model structures we have proposed are consistent with
critical roles for key residues identified by site-directed mutagenesis to be important for MCT1 function and suggest the
6
HALESTRAP
Figure 3. Proposed structure of MCT1. From left to right the structures shown represent the predicted structures of MCT1, derived
by homology modeling, in the closed conformation, the open conformation with the inhibitor DIDS bound and the open conformation in association with the ancillary protein embigin to which it is crosslinked by DIDS. The transition from open to closed conformation involves movement of the C-terminal 6 TM helices relative to the N-terminal 6 TM helices as indicated. Key lysine residues involved in the binding of DIDS and crosslinking to embigin are shown. Diagrams are modified from those in (21) where further details may be found.
following translocation cycle (22). In the ‘‘open’’ conformation,
MCT1 has an essential lysine (K38) in a hydrophobic pocket at
the bottom of a substrate-binding channel. The hydrophobic
environment ensures that this is normally uncharged, but when
it accepts a proton it provides a binding site for the monocarboxylate anion. This induces a domain rearrangement to form
the closed conformation with the lactate and proton being
passed through the channel. This is likely to involve aspartate
and arginine residues (D302/R306) that are present as an ion
pair in the channel and known to be essential for activity from
site-directed mutagenesis (21, 45). As lactic acid bound to K38
is transferred to D302/R306, the K38 is deprotonated again, and
MCT1 relaxes back to the closed state, opening the D302/R306
site to the intracellular medium. This allows the bound lactic
acid to diffuse into the intracellular medium, and the transport
cycle to repeat. Close to the D302/R306 ion pair is phenylalanine 360 that has been identified to play an important role in
determining which substrates are transported by MCT1 (21).
When phenylalanine 360 is mutated to a cysteine, MCT1 can
transport the larger monocarboxylate, mevalonate (12).
Measurement of the Km values of chimeric transporters combining different domains of MCT1 and MCT4 revealed that
both the C- and N-terminal halves of the molecule influence
transport kinetics consistent with our proposed translocation
mechanism that requires Lys38 in TM1 in addition to Asp302
and Arg306 in TM8 (19). Similar studies with MCT1/MCT2
chimaeras suggested that the TM domains, and especially TM7TM12, are the major determinants of L-lactate affinity with the
C-terminus and intracellular loop between TMs 6 and 7 having
little effect (19).
SOME MCTS REQUIRE AN ANCILLARY PROTEIN FOR
PLASMA MEMBRANE EXPRESSION AND ACTIVITY
Our studies on the inhibition of MCT1 in rat erythrocytes by
DIDS showed its binding to be reversible initially but subsequently to become irreversible as one of its two isothiocyano
groups covalently modifies a lysine on MCT1 [see (1)]. However, Western blotting with antibodies against MCT1 revealed
that by using its second isothiocyano group DIDS can also
crosslink MCT1 to another protein of about 70 kDa which we
identified as embigen (also known as gp-70) (44). Embigin is
not expressed in many tissues but basigin (also known as
CD147, OX-47, EMMPRIN, or HT7) is a closely related protein
that is widely expressed. Both basigin and embigin have a single transmembrane domain containing a conserved glutamate
residue, a short intracellular C-terminus, and a large glycosylated extracellular domain with two or three immunoglobulin
domains depending on the splice variant (47). We showed that
when attempts were made to overexpress MCT1 or MCT4 in a
variety of cell lines the proteins failed to reach the plasma
membrane but accumulated in the perinuclear region characteristic of the Golgi apparatus. However, when coexpressed with
basigin both MCT1 and MCT4 were correctly targeted to the
plasma membrane, suggesting that basigin acts as an essential
chaperone for both MCTs (48). The use of a CD2/basigin chimera revealed that it is the transmembrane domain and/or intracellular tail of basigin rather than the extracellular domain that
is required for this role (48).
Immunofluorescence microscopy revealed that MCT1 and
MCT4 colocalize with basigin in a variety of different cells and
tissues implying that the proteins remain associated (48–50),
MCT STRUCTURE AND FUNCTION
and this continued association was confirmed by several techniques. First, MCT1 and basigin could be coimmunoprecipitated
from solubilized plasma membranes (48). Second, inducing
aggregation of basigin to a cap at one end of a cell with a
cross-linking antibody induced caused MCT1 to move to the
same cap (48). Third, constructs of MCT1 containing either the
cyan- or yellow-variants of green fluorescent protein (CFP and
YFP) at the C- or N-terminus were coexpressed at the plasma
membrane with basigin containing either YFP or CFP on the
C-terminus. Using a confocal microscope, we observed fluorescence resonance energy transfer (FRET) between the CFP and
YFP which can only occur when the two proteins are \ 100 Å
apart (51). We were also able to demonstrate FRET between
MCT1-CFP and MCT1-YFP which implied that MCT1 can
form dimers. Taken together with the antibody capping studies,
these data suggest that MCT1 is likely to exist as an MCT1basigin dimer. Subsequent experiments using site-directed mutagenesis and molecular modeling led us to propose that the TM
of the ancillary protein lies adjacent to TMs 3 and 6 of the
MCT (21, 22, 27). We were able to model this interaction (34)
as shown in Fig. 3.
When available, basigin is the preferred binding partner for
MCT1 as confirmed by expression studies in Xenopus oocytes
(19). However, in the absence of basigin, MCT1 will interact
with embigin as it does in rat red blood cells (44). By contrast,
overexpression of MCT2 in the plasma membrane of mammalian cells requires coexpression of embigin rather than basigin
(27) and exogenous expression of MCT2 in Xenopus oocytes is
enhanced by coexpression of exogenous embigin and not basigin (19). These data imply that embigin rather than basigin is
the preferred binding partner for MCT2. However, MCT2 can
interact with basigin if no embigin is available (as in oocytes),
and we have shown that the C-terminus of MCT2 is essential
for this interaction with basigin which is not the case when
MCT2 associates with embigin (19). The C-terminus of MCT1
had no effect on its interaction with either embigin or basigin,
and we have been unable to detect any change in the kinetic
properties of MCT1 when it is associated with embigin rather
than basigin (19). However, inhibition of MCT1 by organomercurial reagents such as pCMBS does require its association with
basigin rather than embigin (27), accounting for the earlier observation that MCT2, which usually associates with embigin,
was insensitive to pCMBs (25). Indeed, we showed that organomercurial agents attack a reactive extracellular cysteine residue
in basigin that is lacking in embigin (19, 27) and the resulting
conformation change in the basigin appears to weaken its interaction the MCT leading to inhibition of transporter activity.
Very recent studies in our laboratory have revealed that inhibition of MCT2 by AR-C155858 was greatly reduced when
MCT2 was expressed in Xenopus oocytes with its preferred
partner embigin, rather than with endogenous basigin, whereas
for MCT1 the choice of ancillary protein was without effect
(19). Use of MCT1/MCT2 chimaeric transporters and MCT1
and MCT2 with their C-terminal tails removed showed that this
7
effect of embigin to modulate MCT2 sensitivity to ARC155858 involves interactions of embigin with both the intracellular C-terminus and TMs 3 and 6 of MCT2 (19).
FUTURE PERSPECTIVES
Considerable progress has been made in characterizing the
properties of different members of the MCT family, and there is
an increasing awareness that MCTs play critical metabolic roles
in a wide range of normal and pathological conditions [see (2,
4, 8)]. However, there remain eight members of the MCT family whose function has yet to be identified. Our working model
of the molecular mechanism of MCT1 suggests key residues
required for proton translocation are not present in MCT 5,
MCT6, MCT9, MCT12, and MCT14. Thus, only MCT 7,
MCT11, and MCT13 are likely to be proton-linked transporters.
Our own unpublished studies have failed to demonstrate protonlinked transport of a wide range of monocarboxylates by MCT7
when expressed in Xenopus oocytes. Clues as to the function of
these orphan members of the MCT family may emerge as disease states are identified that are associated with their mutation.
The recent development by AstraZeneca of a class of potent
MCT1 inhibitors that act as powerful immunosuppressive agents
by inhibiting T-lymphocyte proliferation (17) provides a valuable tool for studying the metabolic roles of MCT1 as well as
illustrating how MCTs may be promising pharmacological targets. Clearly, drugs that are specific and potent inhibitors of the
other MCT isoforms would also be desirable, both for probing
their roles in normal metabolism and as potential therapeutic
agents. Development of such drugs would be greatly enhanced
if three-dimensional structures of the different MCT isoforms
were available and we are currently working on this.
ACKNOWLEDGEMENTS
The author thanks the numerous colleagues who have worked
on MCTs in his laboratory over many years and the many funding bodies who have supported the research. Constraints on the
maximum size of the bibliography means that it has not been
possible to cite directly all authors of the work reviewed.
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9
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