(Received for publication, March 1, 1995; and in revised form, June 19, 1995)
From the
The 24 ANK repeats of the membrane-binding domain of ankyrin
form four folded subdomains of six ANK repeats each. These four repeat
subdomains mediate interactions with at least seven different families
of membrane proteins. In the erythrocyte, the main membrane target of
ankyrin is the Cl/HCO
anion exchanger. This report presents the first evidence that
ankyrin contains two separate binding sites for anion exchanger dimers.
One site utilizes repeat subdomain two (repeats 7-12) while the
other requires both repeat subdomains three and four (repeats
13-24). The two sites are positively coupled with a Hill
coefficient of 1.4. Since the anion exchanger exists as a dimer in the
membrane, the presence of two binding sites on ankyrin allows ankyrin
to interact with four anion exchangers simultaneously. These findings
provide a direct demonstration of the versatility of ANK repeats in
protein recognition, and have important implications for the
organization of ankyrin-linked integral membrane proteins in
erythrocytes as well as other cells.
The spectrin-based membrane skeleton is an interlocking network
of proteins which underlies the plasma membrane. The skeleton was first
identified in erythrocytes, but it is also present under specialized
regions of plasma membrane of cells in many tissues. The membrane
skeleton is comprised of heterotetramers of - and
-spectrin
which form multiple, long range cross-links between cortical actin
filaments. This structure is then linked to the membrane primarily by
ankyrin, which possesses binding sites for
-spectrin and at least
seven membrane proteins(1, 2) .
In human
erythrocytes, the interaction between ankyrin and the cytoplasmic
domain of the Cl/HCO
anion exchanger (Band 3) provides a major linkage between the
spectrin skeleton and the plasma
membrane(3, 4, 5) . This linkage gives the
erythrocyte membrane elastic properties which allows the plasma
membrane to deform without vesiculation. Mutations or reductions in
ankyrin which disrupt the linkage with the anion exchanger decouple the
structural support of the spectrin skeleton from the membrane and
result in severe hemolytic spherocytosis and
anemia(2, 6, 7) .
The anion exchanger binding activity has been localized to the N-terminal, 89-kDa domain of ankyrin(8) . The amino acid sequence of the 89-kDa domain is dominated by a tandem array of 24 ANK repeats: a 33-amino acid motif utilized in a diverse group of proteins for protein recognition(2, 9, 10) . A 43-kDa proteolytic fragment derived from the C-terminal half of the 89-kDa domain is capable of interacting with the anion exchanger with high affinity, indicating that a major site of interaction is localized to repeats 13-24(8) . Activities of a series of deletion constructs demonstrate that repeats 22 and 23 are necessary but not sufficient for high affinity binding of the 43-kDa domain with the anion exchanger(11) . It was not established in this study whether repeats 22 and 23 are required for stable folding or are involved in direct contact with the anion exchanger. Moreover, the 89-kDa domain is more active than the 43-kDa fragment, suggesting that the first 12 repeats of the 89-kDa domain also participate in the interaction with the anion exchanger.
Individual ANK repeats of the 89-kDa domain do not exhibit stable secondary structure; they fold cooperatively into subdomains containing six repeats. In this manner, the 24 repeats of the 89-kDa domain of ankyrin form four subdomains, each containing a specific, ordered group of six repeats(12) . Interaction of the 89-kDa domain with the cytoplasmic domain of the anion exchanger was examined in this study using protein constructs whose boundaries correspond to those of the six-repeat folding units. A major finding from these experiments is the discovery that the 89-kDa domain can simultaneously bind to two anion exchanger dimers through two distinct high affinity binding sites. One site is located on the second subdomain. The other site requires participation of both the third and fourth subdomains. Association of anion exchanger dimers with the 89-kDa domain exhibits positive cooperativity, implying communication between subdomains. Binding of each ankyrin to two anion exchanger dimers provides a biochemical rationale for a 4:1 complex of anion exchanger polypeptides with ankyrin in erythrocyte membranes.
Figure 1: Purification and map of repeat domain constructs. Recombinant protein constructs were designed, based upon the repeat subdomains present in the 89-kDa domain membrane-binding domain of ankyrin (upper panel). Proteins containing one (D1, D2, D3, and D4) and two (D1-D2, D2-D3, and D3-D4) repeat subdomains and native 89-kDa domain containing all four repeat subdomains were uniformly loaded on an SDS-polyacrylamide gel with 1.5 µg of protein/lane. The gel (lower left) was electrophoresed and stained with Coomassie Blue. The name of the protein is used as the designation for each lane. The region contained within each construct is shown in the lower right. The numbers refer to the repeats. N designates the first 9-10 amino acids from the N terminus of ankyrin to the start of the first repeat. H refers to the ``'hinge region,'' a 32-amino acid stretch from the end of the last repeat to the chymotryptic site separating the membrane-binding domain form the spectrin-binding domain. The residues contained within each protein are as follows: D1, 1-204; D2, 205-402; D3, 403-600; D4, 601-827; D1-D2, 1-402; D2-D3, 205-600; D3-D4, 403-827; and the 89-kDa domain, 1-827.
The ability of labeled anion exchanger
cytoplasmic domain (CDB3) to interact with the various
ankyrin-conjugated beads was assessed as follows. 0.8% (w/v) ANK
repeat-conjugated beads were incubated with increasing concentrations
of I-labeled CDB3 in a buffer containing 10 mM sodium phosphate, pH 7.2, 1 mM NaN
, 1
mM dithiothreitol, 0.1% Tween 20, and 10 mg/ml bovine serum
albumin. Assays were performed in triplicate on ice for 3 h with mild
shaking. Bound and free ligand were separated by centrifugation (10,000
g for 15 min) of the reactions over a 10% sucrose
cushion. Nonspecific interactions were determined in parallel assays in
the presence of 2 µM unlabeled CDB3 and were subtracted
from the mean values of the triplicate assays. The interactions were
plotted assuming the anion exchanger was a monomer, since this
represents the total number of potential interaction sites in a given
assay. Since CDB3 is a dimer in solution (15) and the ankyrin
regions are immobilized on a large surface, the concentration of
reactive units is half the concentration of anion exchanger
polypeptides. The affinities reported have been corrected to reflect
the dimeric state of the cytoplasmic domain.
Figure 2:
Hill plot analysis of the interaction of
native 89-kDa domain and construct D3-D4 with stripped erythrocyte
membranes. Membrane binding assays were performed using I-labeled 89-kDa domain (220,000 cpm/pmol) and D3-D4
(33,100 cpm/pmol) with KI stripped inside-out erythrocyte membranes
(see ``Experimental Procedures''). Affinities were calculated
from the zero intercept on the y axis. Construct D3-D4
displayed a linear plot with a K
of 10
nM and a Hill coefficient of 0.95. The 89-kDa domain
interaction had a K
of 1.5 nM,
but had a biphasic Hill plot with a Hill coefficient of 2.2 at
concentrations below the K
and a Hill
coefficient of 0.9 above the K
. In the
Hill plot, [free ligand] is the concentration of free 89-kDa
domain or D3-D4 in solution at each point.
is the fraction of
sites on the membrane occupied by labeled protein.
, 89-kDa
domain;
, D34.
Binding of
CDB3 to immobilized 89-kDa domain (Fig. 3) was cooperative as
evidenced by a concave Scatchard plot (Fig. 3B) and a
Hill coefficient of 1.4 (Fig. 3C). Half-maximal binding
occurred at 20 nM based on the Hill plot. The interaction of
CDB3 with immobilized native ankyrin (protein 2.1) (Fig. 4) was
similar to the interaction with immobilized 89-kDa domain. As with the
89-kDa domain, the interaction with whole ankyrin displayed a slightly
sigmoidal saturation curve. The Scatchard plot was concave indicating
positive cooperativity. The Hill plot quantified the cooperativity with
a Hill coefficient of 1.3. The calculated K of 40 nM for the interaction of CDB3 with ankyrin
was only 2-fold weaker than that of CDB3 with the 89-kDa domain. The
overall similarity between the two interactions support the conclusion
that the 89-kDa domain contains all of the activity of ankyrin for the
interaction with the anion exchanger.
Figure 3:
CDB3 interaction with native ankyrin. The
216-kDa isoform of ankyrin (band 2.1) was purified from erythrocytes
and coupled to latex beads. These beads were assayed for activity as
described under ``Experimental Procedures.'' The saturation
plot of I-labeled CDB3 (6932 cpm/pmol) with the ankyrin
coupled beads is displayed in panel A. Concentration
(nM) is the concentration of CDB3 polypeptide. Bound
(fmol) is the femtomoles of CDB3 bound to the latex beads. Panel B contains the corresponding Scatchard plot and shows a
partially concave interaction. Bound (fmol) is again the
femtomoles of CDB3 bound. Bound/Free (fmol/nM) is the
ratio between the femtomoles of CDB3 bound versus the
nanomolar concentration of CDB3 free in solution. The Hill plot in panel C indicates that the interaction with native ankyrin has
positive cooperativity with a Hill coefficient of
1.3.
Figure 4:
The
CDB3 interaction with the complete set of four repeat domains (89-kDa
domain). The interaction of the cytoplasmic domain of the erythrocyte
anion exchanger (CDB3) with the membrane-binding domain containing all
four repeat domains (89-kDa domain) was assessed by coupling the 89-kDa
domain region to latex beads and measuring the affinity of I-labeled CDB3 (5136 cpm/pmol) for these beads (see
``Experimental Procedures''). The saturation plot in panel A shows the interaction of CDB3 with immobilized 89-kDa
domain as a function of increasing concentrations of CDB3 polypeptide.
The Scatchard plot in panel B is concave implying a positively
cooperative interaction. Cooperativity was quantitated in the Hill plot (panel C) which gave a Hill coefficient of 1.4. A K
of 20 nM for the CDB3 dimer
was calculated from the zero intercept of the y axis on the
Hill plot.
Construct D3-D4 exhibited half-maximal binding at 40 nM, but did not display the positively cooperative interaction of the 89-kDa domain (Fig. 5) (Hill plot not shown). The issue of whether the binding site present on construct D3-D4 required both subdomains three and four was addressed by evaluating binding activity of the individual subdomains (Fig. 5). Neither subdomains three nor four were active alone, indicating that both subdomains are required for the interaction with the anion exchanger (Fig. 5).
Figure 5:
Activity of constructs D3, D4, and D3-D4.
Repeat domain constructs D3, D4, and D3-D4 were coupled to latex beads
and the affinity of I-labeled CDB3 (5136 cpm/pmol) for
these beads were measured as described under ``Experimental
Procedures.`` Panel A shows the saturation plot for the
interaction of labeled CDB3 with D3, D4, and D3-D4 coupled beads.
, D3;
, D4;
, D34. Panel B contains the
Scatchard plot for the interaction with the D3-D4 beads. An affinity of
45 nM was calculated from the slope of the Scatchard
plot.
The positive cooperativity under the assay conditions of Fig. 3and Fig. 4implies that ankyrin and the 89-kDa domain contain multiple binding sites for anion exchanger dimers. Since construct D3-D4 lacks cooperativity, the N-terminal subdomains of ankyrin provide either a second site for the anion exchanger or are indirectly responsible for the positive cooperativity.
Figure 6:
CDB3
interaction with constructs D1-D2 and D2-D3. The affinity of I-labeled CDB3 for D1-D2 and D2-D3 coupled beads was
assessed as described under ``Experimental Procedures.'' Panel A shows a saturation plot of the interaction of labeled
CDB3 (5136 cpm/pmol) with immobilized D1-D2. Panel B contains
the saturation plot of labeled CDB3 with immobilized D2-D3.
Interactions with both D1-D2 and D2-D3 displayed biphasic Scatchard
plots (panels C and D, respectively). A K
of 38 nM for the D1-D2 beads
was calculated from the high affinity interaction seen in the Scatchard
plot in panel C. A K
of 10
nM for construct D2-D3 was calculated from the Scatchard plot
in panel D.
Subdomain two is common between D1-D2 and D2-D3
constructs and this domain alone proved to be responsible for the
N-terminal second binding site (see Fig. 7). Construct D2 had a K of 12 nM and a biphasic
Scatchard plot virtually identical to that of construct D2-D3. Since
subdomains one and three alone had no activity and the interaction of
D2 was approximately as strong as construct D2-D3, the second site
activity is likely to be completely contained in subdomain two. In this
case, construct D1-D2 would be expected to have an affinity similar to
that of construct D2. The lower affinity of D1-D2 may be the result of
the oligomerization state of this construct. Even in 1 M NaBr,
construct D1-D2 behaves as a dimer (12) and this may adversely
affect the ability of construct D1-D2 to interact with the anion
exchanger.
Figure 7:
CDB3 only interacts with D2. Domains one
(D1), two (D2), and three (D3) were coupled to latex beads and assayed
for the ability to interact with I-labeled CDB3 (5136
cpm/pmol) as described under ``Experimental Procedures.'' Panel A contains the saturation plots of these assays.
, D1;
, D2;
, D3. Panel B contains the
Scatchard plot for the interaction with D2 coupled beads. A 12
nM affinity was calculated from the slope of high affinity
interaction in the Scatchard plot.
The biphasic Scatchard plots seen with constructs D2, D1-D2, and D2-D3 result either from negative cooperativity or high and low affinity sites. One possibility is that subdomain two contains a site for each polypeptide chain in the anion exchanger dimer. At moderate concentrations of dimer, both sites are used to interact with one dimer. At high concentrations of dimer, a free dimer may compete with the bound dimer for one of the sites on subdomain two. This competition may be unfavorable and thereby cause negative cooperativity. Another possibility is that there is a second site on subdomain two for the anion exchanger. This weaker interaction may be a partial site which may participate in the D3-D4 interaction and thereby contribute to the positive cooperativity seen in the complete 89-kDa domain region.
The physically distinct anion exchanger binding sites on subdomain two and on subdomains three and four were compared in terms of their sensitivity to ionic strength. The interaction between CDB3 and construct D2-D3 was completely abolished by concentrations of NaCl above 0.5 M while the interaction with D3-D4 was unaffected at concentrations as high as 1.5 M (Fig. 8). These results suggest that electrostatic interactions mediate the association with subdomain two while non-ionic contacts mediate the interaction with subdomains three and four. 1.5 M NaCl only inhibited the interaction with the native 89-kDa domain by 40-50%. These results indicate that the 89-kDa domain has two sites, one NaCl-sensitive and one insensitive. Of interest is the finding that half-maximal inhibition of binding to construct D2-D3 occurred at 0.17 M NaCl, while binding to the 89-kDa domain had a half-maximal inhibition at 0.5 M NaCl. The difference may be the result of cooperativity between the D3-D4 site and the D2 site.
Figure 8:
NaCl sensitivity of the interaction
between CDB3 and constructs D2-D3, D34, and native 89-kDa domain.
Assays were performed using 20 nMI-labeled CDB3
(6932 cpm/pmol) in the presence of increasing concentrations of NaCl
with beads coated with construct D2-D3 (panel A), D3-D4 (panel B), or whole 89-kDa domain (panel C). The
half-maximal competition for D2-D3 bead interaction was 175
mM. The D3-D4 bead interaction could not be significantly
competed with NaCl even at the highest concentrations of NaCl (1.6 M). The interaction with the 89-kDa domain coupled beads was
competed 40-50% by increasing concentrations of NaCl. The
half-maximal competition for the 89-kDa domain beads was 500 mM NaCl.
Figure 9:
89-kDa domain dependent interaction of
CDB3 with immobilized CDB3. Increasing concentrations of I-labeled CDB3 (63, 200 cpm/pmol) were incubated with
immobilized CDB3 in the presence or absence of 100 nM 89-kDa
domain. Labeled CDB3 did not display saturable binding to the
immobilized CDB3 in the absence of 89-kDa domain. Displayed is the
difference plot of the interaction in the presence of the 89-kDa domain
subtracted from the interaction in the
absence.
This paper presents the first evidence that ankyrin contains two separate binding sites for anion exchanger dimers. One site is located on repeat subdomain two; the other site requires both repeat subdomains three and four. The two sites can be distinguished by their different affinities and sensitivities to high ionic strength. The two sites are positively coupled with a Hill coefficient of 1.4. Since the interaction with D2 has a 4-fold higher affinity than that with the D3-D4, repeat domain two may contain the primary site of interaction with the anion exchanger. These findings provide a direct demonstration of the versatility of ANK repeats in protein recognition, and have important implications for the organization of ankyrin-linked integral membrane proteins in erythrocytes as well as other cells.
The
ability of ankyrin to bind to two anion exchanger dimers helps resolve
an unexplained discrepancy between the number of anion exchangers and
the capacity of ankyrin-depleted erythrocyte membranes to rebind
ankyrin. Each erythrocyte contains roughly 10 anion
exchangers, assembled into 4
10
intramembrane
particles comprised primarily of dimers as well as a smaller number of
higher oligomers(16, 17, 18) . Anion
exchanger dimers can associate with ankyrin with high affinity in
solution(4, 5, 19) , and all anion exchangers
appear to be functionally and structurally
equivalent(3, 5, 19, 20) .
Nevertheless, ankyrin-depleted erythrocyte membranes can rebind a
maximum of only 100,000-250,000 ankyrin molecules(4, 21) corresponding to a limiting stoichiometry of approximately
one ankyrin for four anion exchanger polypeptides. The evidence
presented in this study indicates that ankyrin contains two binding
sites for anion exchanger dimers and readily explains how four anion
exchangers can interact with one ankyrin molecule. The limited number
of binding sites for ankyrin can be explained if anion exchanger dimers
in the membrane preferentially interact with an ankyrin-anion exchanger
dimer complex over ankyrin free in solution. The cooperativity of the
CDB3 interaction with the 89-kDa domain and whole ankyrin ( Fig. 3and Fig. 4) provide a mechanism for this
selectivity.
A higher order complex of anion exchanger with ankyrin
in intact erythrocyte membranes has also been predicted by rotational
diffusion studies(22) . Diffusion measurements of the anion
exchanger indicate that 40% of the anion exchangers (4 10
per cell) display restricted rotational diffusion(22) .
Ankyrin is present in approximately 10
copies per
cell(23) . Thus, assuming the immobile anion exchangers are
directly coupled to ankyrin, four anion exchangers are bound to every
ankyrin. The formation of this complex can be directly and simply
explained by the presence of two sites on ankyrin for the anion
exchanger dimer.
In the interaction with erythrocyte membranes, the 89-kDa membrane-binding domain of ankyrin exhibits positive cooperativity, while whole ankyrin exhibits negative cooperativity (8) (Fig. 2). When the assay is reversed and the anion exchanger is the saturating ligand, interactions with both the 89-kDa domain and ankyrin are positively cooperative ( Fig. 3and Fig. 4). Positive cooperativity implies that the interaction of one ligand promotes the interaction of additional ligands. Thus, in membrane binding assays with the 89-kDa domain, the interaction of one 89-kDa domain promotes the interaction of a second 89-kDa domain to the same anion exchanger dimer. The two sites of interaction on the anion exchanger dimer are separate and not within steric hinderance of each other. In the case of ankyrin, the interaction with erythrocyte membranes has negative cooperativity, indicating that interaction of one ankyrin inhibits the interaction of a second ankyrin to the same anion exchanger dimer. Ankyrin is a much larger molecule with a spectrin-binding domain and C-terminal region which may inhibit access to the second site on the anion exchanger dimer. When the 89-kDa domain or ankyrin is immobilized on latex beads this steric hindrance no longer applies; sites on the 89-kDa domain and ankyrin are being filled and steric hindrance between anion exchanger dimers will determine access to the sites (see Fig. 10and 11).
Figure 10: The four repeat domains of the membrane-binding domain contain two sites for the anion exchanger. The 89-kDa domain region contains 24 repeats organized into four folding domains. These four domains form two binding sites for the anion exchanger. Domain two contains one site. The other site requires both repeat domains three and four. The D2 site is salt sensitive, suggesting that this interaction is mediated by electrostatic interactions. The site on D3-D4 is not salt sensitive, indicating that this interaction is likely mediated by hydrogen bonding or hydrophobic contacts.
The difference in salt sensitivities of ankyrin constructs D2-D3 and D3-D4 (Fig. 8) supports the presence of two separate ankyrin binding sites on anion exchanger dimers which differ in their dependence on electrostatic bonds. The interaction with the D3-D4 site is not salt sensitive, suggesting that this interaction is mediated by non-ionic contacts such as hydrogen bonding or hydrophobic surfaces. The salt insensitivity of the D3-D4 site is consistent with the stability of the ankyrin association with erythrocyte membranes at high concentrations of salt. In contrast, the subdomain two site was sensitive to high salt, suggesting that the interaction with the subdomain site two involved electrostatic contacts. Localization studies of the ankyrin site on the anion exchanger have identified two regions involved in the interaction(24, 25) . One region was localized to the acidic N terminus; the other, to the region surrounding the flexible hinge. The highly charged nature of the N terminus suggests that this region may contain the site for subdomain two. If this is the case, then the D3-D4 site may involve the region surrounding the hinge (Fig. 10).
An alternative form of the anion exchanger missing the N-terminal 79 residues is expressed in kidney intercalated cells and has recently been shown to lack the ability to bind to construct D3-D4(26) . This result suggests that the D3-D4 site on the anion exchanger requires the first 79 amino acids. One possibility is that since both subdomains three and four are required for the D3-D4 interaction with the anion exchanger, one subdomain interacts with the first 79 amino acids and the other interacts with the hinge region. The location of the subdomain two site on the anion exchanger has not yet been evaluated. If this site also requires the N-terminal 79 amino acids, the truncated form of the anion exchanger may not interact with ankyrin. A lack of cytoskeletal attachment may allow for rapid turnover in the level of this truncated form of the anion exchanger in kidney membranes.
The interaction of ankyrin with the anion exchanger is regulated by alternative splicing and phosphorylation. An alternatively spliced form of erythrocyte ankyrin, which removes a portion of the C-terminal region, exhibits increased capacity and higher affinity for the anion exchanger in erythrocyte membranes(27, 28) . Both ankyrin and the cytoplasmic domain of the anion exchanger are substrates for protein kinase A and casein kinase. Phosphorylation by these kinases affect the capacity of the interaction of ankyrin with the cytoplasmic domain of the anion exchanger(29, 30) . It will be of interest to define these regulatory effects in terms of the ankyrin-anion exchanger contacts resolved in this study.
Several predictions can be made
from the finding of two distinct binding sites on both ankyrin and the
anion exchanger. In erythrocyte membranes, the anion exchanger dimer is
present at a 4-5-fold higher concentration than ankyrin.
Deficiencies in the anion exchanger which reduce the ratio between the
anion exchanger and ankyrin may allow two ankyrins to assemble on
individual anion exchanger dimers. Since ankyrin is also bivalent, this
interaction may lead to aggregation through the formation of
polymers. This
process may occur naturally when hemichromes interact with the anion
exchangers to form insoluble complexes(31) , thereby reducing
the level of free dimer in the membrane. Another route to this
aggregation may be caused by denaturation or oxidation of the anion
exchanger which may promote the formation of tetramers. Since ankyrin
has two sites for the anion exchanger, such a situation could result in
a [(AE1)
-ANK]
polymer. These
polymerization events may be part of the senescence process in which
the erythrocyte membrane becomes more rigid. Several clinical
conditions have been described wherein alterations in anion exchager
result in abnormal membrane properties. These include Melanesian
ovalocytosis which is caused by a 9-amino acid deletion in the anion
exchanger (32, 33) and a form of hereditary
spherocytosis caused by reduced levels of the anion
exchanger(34) .
Interaction of the anion exchanger with ankyrin utilizes three of the four repeat subdomains, yet the anion exchanger is only one of seven known ankyrin-binding membrane proteins. How then are sites for these seven targets accommodated on the repeat domains and how did they evolve? The simple hypothesis that the repeat domains are recognizing a common feature on the various targets is unlikely since the seven different families do not contain areas of homology in their cytoplasmic domains. While the possibility that repeat subdomains recognize a common tertiary structure cannot be discounted, a more likely hypothesis is that membrane proteins generated unique interactions with ankyrin through convergent evolution.
This evolved fit model (35) for the development
of ankyrin interactions makes several predictions. First, if the
membrane proteins evolved interactions with ankyrin, sites should not
be restricted to individual repeat domains. This has been shown here
with the site on D3-D4 for the anion exchanger. In addition, the
Na/K
-ATPase requires both 89-kDa
domain and the spectrin-binding domain for its interaction with
ankyrin(35) . Second, because the anion exchanger utilizes
three of the four repeat domains, the sites for other membrane proteins
will likely overlap with the sites for the anion exchanger. This
overlap may result in steric competition between different membrane
proteins for sites on ankyrin. Currently known members of the ankyrin
gene family, ankyrin
, ankyrin
, and
ankyrin
, contain conserved, but not identical
membrane-binding
domains(36, 37, 38, 39) . The
conservation is strong enough that the three membrane-binding domains
will likely interact with the complete set of ankyrin binding membrane
proteins. However, the sequence differences may alter the relative
affinities of these interactions. In the case of the anion exchanger, a
membrane-binding domain construct of ankyrin
is 8-fold
weaker than the D3-D4 region of ankyrin
in membrane binding
assays(11) . Differences in relative affinity may allow
selective sorting of ankyrin isoforms with their appropriate membrane
proteins.
Future work will hopefully elucidate the physiological role for multiple sites for the anion exchanger on ankyrin. An interesting question is whether the interaction of ankyrin with the anion exchanger precludes interactions with other ankyrin binding membrane proteins or whether ankyrin can assemble a complex of membrane proteins under specialized membrane domains.
Addendum-While this work was in review, electron microscopy has observed ankyrin dependent aggregation of the anion exchanger into particles of a size consistent with a complex containing two anion exchanger dimers(40) . This observation confirms our biochemical evidence which indicates that ankyrin can form a 4:1 complex with the anion exchanger through the use of two distinct sites in the membrane-binding domain.