(Received for publication, August 3, 1995; and in revised form, September 26, 1995)
From the
Cytoskeleton membrane associations are important for a variety
of cellular functions. The anion exchanger of erythrocytes (AE1) and
Na,K
-ATPase of polarized epithelial
cells provide well studied examples of how integral membrane proteins
are anchored via the linker molecule ankyrin to the spectrin-based
membrane cytoskeleton. In the present study we have generated several
recombinant fragments of the large (third) cytoplasmic domain (CD3) of
Na
,K
-ATPase to define binding sites
of ankyrin on CD3 at a molecular level. We provide evidence that a
cluster of four amino acids, ALLK, is essential for binding of ankyrin
to both recombinant CD3 and to native
Na
,K
-ATPase. Once bound,
conformational changes might uncover further binding sites for ankyrin
on Na
,K
-ATPase. A motif related to
the ALLK cluster is also present in the cytoplasmic domain of AE1 where
this sequence (ALLLK) turned out to be also important for ankyrin
binding. These motifs are highly conserved during evolution of both
Na
,K
-ATPase and AE1, further
underlining their potential role in cytoskeleton to membrane linkage.
Cellular differentiation and several cellular functions depend
to a large degree on the compartmentalization of particular membrane
proteins such as receptors, adhesion molecules or ion translocating
proteins to specialized domains of the cell surface (for review, see (1, 2, 3) ). One mechanism of how certain
membrane proteins are placed at specialized sites of the plasma
membrane could be by linkage of their cytoplasmic domains to the
cytoskeleton. The HCO,
Cl
-exchanger of erythrocytes (anion exchanger 1, AE1) (
)and the sodium pump
(Na
,K
-ATPase) of transporting
epithelia provide two rather well studied examples of how
ion-translocating integral membrane proteins are tethered via specific
linker molecules to the fibrous scaffold of spectrin and actin that
extends underneath the plasma membrane of virtually all cell types of
the body (for review, see (1, 2, 3) ).
Ankyrin is the main linker molecule that connects the cytoplasmic
domains of both AE1 and Na
,K
-ATPase
to
-spectrin of the membrane
scaffold(4, 5, 6, 7) .
In
transporting epithelia interaction of
Na,K
-ATPase and AE1 with the
spectrin-based membrane scaffold is considered important for the
polarized restriction of these transporters to either the apical or the
basolateral cell
surface(5, 8, 9, 10, 11, 12) .
Polarity of the Na
,K
-ATPase has
profound implications for the direction of the transport of sodium and
several other ions and molecules across the epithelial layer. In
various transporting epithelia (such as kidney tubules, parotid gland,
retinal pigment epithelium, choroid plexus, Mardin Darby canine kidney
(MDCK) cell line) Na
,K
-ATPase is
codistributed with ankyrin and can be copurified (coimmunoprecipitated)
with ankyrin and
spectrin(5, 9, 10, 11, 12, 13) .
Binding of erythrocyte ankyrin to kidney
Na
,K
-ATPase in vitro(4, 5, 14) and competitive inhibition of
this interaction by addition of the cytoplasmic domain of
AE1(5, 13) suggests that there might be a common
binding site on ankyrin involved in binding to both AE1 and
Na
,K
-ATPase. Binding of ankyrin
fragments to the
-subunit of
Na
,K
-ATPase indicated the involvement
of the AE1-binding domain of ankyrin and, in addition, a further domain
of ankyrin not involved in AE1 binding(6) . These observations
are compatible with a recent report describing binding of erythrocyte
and MDCK ankyrin to two of the five putative cytoplasmic domains of
Na
,K
-ATPase, i.e. to
cytoplasmic domains 2 and 3(14) .
The present study was
performed to obtain more detailed information about the ankyrin binding
sites on Na,K
-ATPase, if possible at
the amino acid level. We confined this study to the large cytoplasmic
domain (CD3) because we found in a screening approach that a
recombinant protein containing a portion of CD3 blocked binding of
erythrocyte ankyrin to native kidney
Na
,K
-ATPase. The most striking
outcome of this study is that a motif of four amino acid residues
(ALLK) appears to be essential for ankyrin binding. A similar motif
(ALLLK) occurs in the sequence of the cytoplasmic domain of AE1 where
it appears to participate also in ankyrin binding.
Polymerase chain reaction amplification
(20 cycles) was conducted under the following conditions: 0.9 pM plasmid DNA, 0.4 pM of each primer, 0.4 mM of
each dNTP, 1 unit of Taq polymerase (Boehringer Mannheim), 50
mM KCl, 1 mM MgCl adjusted to pH 8 (at
room temperature, RT) by 10 mM Tris/HCl. Denaturation was
performed at 94 °C (1 min), hybridization (2 min) 2 °C below
the melting temperature of the primers (18) and polymerization
at 72 °C (2 min).
Purity of the preparation was assayed by SDS-PAGE (10%). Removal of
fusion peptides was performed by cleavage with enterokinase (23) that requires 10 mM CaCl for full
activity. After dialysis of fusion proteins (concentration of up to 120
µg/ml) with 10 mM CaCl
in 10 mM Tris/HCl (pH 8 at RT), 12.5 µg/ml enterokinase (Boehringer
Mannheim) was added, and cleavage was allowed to proceed for
12-20 h at 37 °C.
Figure 1:
Position and electrophoretic
(SDS-PAGE) characterization of recombinant fragments of CD3 of rat
Na,K
-ATPase (
1-subunit) used for
binding studies. Numbering of amino acid residues follows Herrera et al.(15) . CD3-7* carries a point mutation in which
lysine 458 was exchanged against glutamic acid. SDS-PAGE of purified
pig kidney Na
,K
-ATPase and human
erythrocyte ankyrin is also shown.
In some binding
studies biotinylated ankyrin (0.5 µg/ml
2.5
10
M) was preincubated for 5 h at
4 °C with each of the following peptides at a concentration of
10
M: ALLK (CD3 of
Na
,K
-ATPase), LRALLLKHSH (cytoplasmic
domain of AE1) LAKL (nonsense control), WAGARPTLGP (control, sequence
of the exoplasmic ``Z-loop'' of anion exchanger 2). Peptides
were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)
method and purified by reverse phase high performance liquid
chromatography(28) .
In a further
cosedimentation assay recombinant fragment 7 of the cytoplasmic domain
3 of Na,K
-ATPase (CD3-7) and its
mutated counterpart (CD3-7*), respectively, were added at a
concentration of 18 µg/ml to 1 µg/ml biotinylated ankyrin in
PBS, 5% BSA. After 30 min at 37 °C this mixture was used for
binding studies with Na
,K
-ATPase
vesicles as described above.
Localization and electrophoretic characterization of
recombinant fragments of Na,K
-ATPase
and other proteins used for binding studies are documented in Fig. 1.
Specific binding of ankyrin to the large (third)
cytoplasmic domain (CD3) of
Na,K
-ATPase was shown by dot blot
assay in which a bacterially expressed large portion of CD3 (CD3-1,
that covers almost the entire length of CD3) (Fig. 1) was
immobilized on nitrocellulose (solid phase) and incubated with
biotinylated ankyrin (soluble phase). No binding of ankyrin was
observed to BSA, soluble bacterial proteins, a bacterially expressed
ankyrin fragment carrying the same short fusion peptide as CD3-1, and
the fusion peptide itself (Fig. 2).
Figure 2: Dot blot assay demonstrating specific binding of biotinylated ankyrin (0.5 µg/ml) to 10 µg of immobilized CD3-1. (1) No significant binding of ankyrin is detectable to the following control proteins/peptides (10 µg per dot); (2) BSA; (3) recombinant fragment of human brain ankyrin; (4) soluble bacterial proteins; and (5) fusion peptides.
To further identify the
binding site of ankyrin on CD3-1, six fragments of CD3-1 were generated
(CD3-2 to CD3-7) and subjected to dot blot assay using the same
protocol. Ankyrin bound to CD3 fragments 2, 3, and 7, but not to CD3
fragments 4, 5, and 6 (compare Fig. 3with Fig. 1). After
removal of the fusion portion, binding occurred also to CD3-4
(abbreviated as CD3-4) but not to
CD3-5, indicating that the
19 amino acid residues 447-465 of
Na
,K
-ATPase contain a site essential
for ankyrin binding (see also ``Discussion'').
Figure 3:
Binding of biotinylated ankyrin to various
recombinant fragments of CD3 (compare with Fig. 1). No binding
occurs to CD3-4, CD3-5, and CD3-6. However, after removal of the fusion
peptide by enterokinase cleavage (CD3-4,
CD3-5) binding is
seen to
CD3-4 but not to
CD3-5, indicating that the binding
site for ankyrin on CD3 is located between amino acid residues 447 and
465.
Comparison
of this sequence stretch (VAGDASESALLKCIEVCCG) with the sequences of
rat, mouse, and human erythrocyte AE1 (30) revealed a short
common motif (ALLK/ALLLK) shared by the cytoplasmic domains of both
Na,K
-ATPase and AE1. To test the
possibility that the ALLK motif of
Na
,K
-ATPase is essential for ankyrin
binding we generated a point-mutated variant of CD3 fragment 7
(CD3-7*), in which lysine 458 (K) was replaced by glutamic acid (E). As
shown in Fig. 4, biotinylated ankyrin bound to nonmutated CD3-7,
but not to CD3-7* in which ALLK was mutated to ALLE.
Figure 4: Dot blot assay to test binding of biotinylated ankyrin to mutated CD3-7 (CD3-7*). In CD3-7* lysine 458 was replaced by glutamic acid. Ankyrin binds to CD3-7, but not to CD3-7*, suggesting that lysine 458 is essential for binding.
The
significance of this observation was further tested by a competitive
binding assay where binding of ankyrin to native kidney
Na,K
-ATPase was tested in the absence
and presence of CD3-7 and CD3-7*, respectively. As expected, binding
(cosedimentation) of ankyrin to
Na
,K
-ATPase vesicles was inhibited by
addition of nonmutated CD3-7, but was not inhibited by the mutated
variant CD3-7* (Fig. 5). These experiments show that the
N-terminal third of cytoplasmic domain 3 of
Na
,K
-ATPase (CD3-7) serves as binding
site for ankyrin and that the ALLK motif within this fragment appears
to be essential for ankyrin binding.
Figure 5:
Cosedimentation of biotinylated ankyrin (1
µg/ml) with pig kidney
Na,K
-ATPase vesicles (40 µg/ml)
in the absence or presence of 18 µg/ml CD3-7 and CD3-7*,
respectively. Pellets were dotted onto nitrocellulose filters and
stained for biotinylated ankyrin. Binding (cosedimentation) of ankyrin
to Na
,K
-ATPase vesicles is inhibited
by CD3-7, but not by the point mutated CD3-7* variant, in which lysine
458 was exchanged against glutamic acid.
In a further series of
experiments we addressed the question whether the ALLK motif is
directly involved in ankyrin binding or whether this motif plays an
indirect role in affecting the conformation of the cytoplasmic domain.
As shown in Fig. 6, the ALLK peptide inhibited binding of
ankyrin to immobilized CD3-7, indicating that the ALLK motif is
directly involved in binding of ankyrin to this portion of the
cytoplasmic domain of Na,K
-ATPase. To
determine whether the ALLLK cluster in the cytoplasmic domain of the
erythrocyte AE1 is also essential for ankyrin binding, we extended
binding studies to the purified cytoplasmic domain of AE1 (CD-AE1).
Since the ALLLK peptide was too hydrophobic (hardly soluble), we used
the extended AE1 sequence LRALLLKHSH. Both peptides (ALLK and
LRALLLKHSH) inhibited binding of ankyrin to immobilized CD-AE1 and also
to CD3-7 of Na
,K
-ATPase. Nonsense and
control peptides did not interfere with binding of ankyrin to either
the CD-AE1 or CD3-7. This indicates that the ALL(L)K clusters on both
Na
,K
-ATPase and AE1 participate
directly in ankyrin binding.
Figure 6:
Influence of the ALLK motif on binding of
ankyrin to the cytoplasmic domains of
Na,K
-ATPase (fragment CD3-7) and of
the erythrocyte anion exchanger (CD-AE1). Binding of biotinylated
ankyrin (0.5 µg/ml
2.5
10
M)
to 10 µg/dot of immobilized CD3-7, CD-AE1, and BSA was assayed in
the absence and presence of the following peptides (10
M): ALLK (potential ankyrin binding motif on CD3-7 of
Na
,K
-ATPase), LRALLLKHSH (sequence of
CD-AE1 containing the related motif ALLLK), LAKL (nonsense peptide as
control for ALLK), and WAGARPTLGP (control peptide, portion of the
exoplasmic Z-loop of anion exchanger 2). The ALL(L)K motifs of
Na
,K
-ATPase and AE1 inhibit binding
of ankyrin to both CD3-7 of
Na
,K
-ATPase and the cytoplasmic
domain of AE1.
Interactions of integral membrane proteins with components of
the cytoskeleton are probably important for diverse cellular functions,
as for example for the assembly of specific plasmalemmal adhesion
domains and for the generation or maintenance of cellular polarity (1, 2, 3, 31) . The erythrocyte AE1
and kidney Na,K
-ATPase provide well
studied examples of such cytoskeleton-membrane associations. Both
integral membrane proteins are linked via ankyrin to the spectrin-based
membrane cytoskeleton. The main ankyrin binding site on AE1 appears to
be located in the midportion of the cytoplasmic
domain(7, 32) . In
Na
,K
-ATPase, cytoplasmic loops 2 and
3 (CD2, CD3) have been implicated in ankyrin binding (14) .
In the present study we were able to identify a main binding site
for ankyrin on CD3 of Na,K
-ATPase.
Generation of various CD3 fragments expressed by E. coli narrowed the protein binding site of ankyrin to a stretch of 19
amino acids (VAGDASESALLKCIEVCCG). This stretch contains the only site
of homology between Na
,K
-ATPase and
the cytoplasmic domain of AE1, that consists of the cluster ALLK
(Na
,K
-ATPase) or ALLLK
(AE1)(5, 13) . Several lines of evidence were provided
indicating that the ALLK motif is essential for binding of ankyrin to
Na
,K
-ATPase (Fig. 7): (a) no ankyrin binding was obtained with those CD3-fragments
of Na
,K
-ATPase that lack the ALLK
motif; (b) mutation of ALLK to ALLE in the CD3-7 fragment
(CD3-7*) abolished binding of ankyrin; (c) the nonmutated
CD3-7 fragment inhibited binding (cosedimentation) of ankyrin to native
Na
,K
-ATPase, whereas the mutated
analogue (CD3-7*) did not interfere with ankyrin binding; and (e) both the ALLK peptide of
Na
,K
-ATPase and the ALLLK peptide of
AE1 inhibited binding of ankyrin to CD3-7, whereas nonsense peptides
(LAKL, WAGHRPTLGP) did not interfere with ankyrin binding.
Figure 7:
Diagrammatic summary of the experimental
background for the main conclusion of this study: the motif ALLK on CD3
of Na,K
-ATPase
-subunit appears
to be essential for ankyrin binding. Binding of ankyrin to the ALLK
motif on CD3 might uncover further binding sites located on
CD2.
Further
support for the involvement of ALLK in ankyrin binding to
Na,K
-ATPase came from experiments in
which CD3-4 was used for binding studies. In this CD3 portion the ALLK
motif is located very close to the N terminus (VAGDASESALLK . . .).
Binding of ankyrin to CD3-4 occurred only when the fusion peptide was
removed by enterokinase cleavage (
CD3-4) (Fig. 3), whereas
in all other CD3 fragments the fusion portion had no effect on ankyrin
binding. The fusion peptide alone did also not interfere with ankyrin
binding. The inability of the uncleaved CD3-4 fusion protein to bind
ankyrin can be tentatively interpreted by steric hindrance caused by
the close proximity between the ALLK motif and the polyhistidine
containing fusion portion.
The ALLK/ALLLK motif turned out to be also essential for binding of ankyrin to the purified cytoplasmic domain of AE1 as indicated by inhibition of ankyrin binding to CD-AE1 in the presence of both ALLK and the ALLLK-containing peptide LRALLLKHSH.
Taken together, these data indicate that the ALLK/ALLLK
clusters of the cytoplasmic domains of
Na,K
-ATPase and AE1, respectively,
are directly involved in ankyrin binding. This notion is further
supported by sequence data showing that the ALL(L)K motif of AE1 is an
evolutionary highly conserved part in all vertebrates studied so far
(fish, chicken, and all mammalian
species)(30, 33, 34, 35) . The same
holds true for the ALLK motif of
Na
,K
-ATPase which has been conserved
without any change from Drosophila to humans (15, 36, 37, 38, 39, 40) .
On the basis of these data it appears that the ALLK motif on
Na,K
-ATPase serves as a primary
docking site for ankyrin. Once bound, conformational changes within CD3
might uncover further binding sites located on the second cytoplasmic
domain of Na
,K
-ATPase(14) .
This model for ankyrin binding to
Na
,K
-ATPase is also compatible with
the observation that ankyrin can bind to
Na
,K
-ATPase with both the AE1-binding
and spectrin-binding fragments(6) , and that two different
portions on AE1 appear to crosstalk in ankyrin binding(32) .
In view of the location of the ALLK motif in close proximity to the
ATPase center of Na,K
-ATPase (41) it is tempting to speculate that such complex interactions
between ankyrin and Na
,K
-ATPase might
be somehow regulated by the activity of the pump, in that the active
pump might fully expose the ALLK motif thereby facilitating its
attachment to the membrane cytoskeleton and its exposure at the cell
surface. The inactive (dead) pump, on the other hand, might no longer
expose the ALLK motif and hence might become prone to cytoskeleton
detachment and removal from the cell surface. Such a mechanism might
explain the low half-life of apically delivered inactive
Na
,K
-ATPase in MDCK cells (42) .