(Received for publication, February 21, 1995; and in revised form, April 25, 1995)
From the
Adducin binds to spectrin-actin complexes, promotes association
of spectrin with actin, and is subject to regulation by calmodulin as
well as protein kinases A and C. Adducin is a heteromer comprised of
homologous - and
-subunits with an NH
-terminal
protease-resistant head domain, connected by a neck region to a
COOH-terminal hydrophilic, protease-sensitive region. This study
provides evidence that adducin in solution is a mixture of heterodimers
and tetramers. CD spectroscopy of COOH-terminal domains of
- and
-adducin bacterial recombinants provides direct evidence for an
unstructured random coil configuration. Cross-linking, proteolysis, and
blot-binding experiments suggest a model for the adducin tetramer in
which four head domains contact one another to form a globular core
with extended interacting
- and
-adducin tails. The site for
binding to spectrin-actin complexes on adducin was identified as the
COOH-terminal tail of both the
- and
-adducin subunits. The
capacity of native adducin to recruit spectrin to actin filaments is
similar to that of adducin tail domains. Thus, adducin tail domains
alone are sufficient to interact with F-actin and a single spectrin and
to recruit additional spectrin molecules to the ternary complex.
The spectrin-based membrane skeleton was first isolated as a meshwork of integral and peripheral membrane proteins when erythrocytes were extracted with non-ionic detergents(1) . Electron micrographs of the stretched membrane skeleton reveal a regular lattice-like organization of 5-6 spectrin molecules linked in a polygonal array (2) . The vertices of this lattice are formed by short actin filaments of 12-14 monomers in length. Accessory proteins are found at the junctions where spectrin and actin interact: these include adducin, protein 4.1, dematin (protein 4.9), tropomyosin, and tropomodulin. These proteins are thought to be important in stabilizing spectrin-actin complexes, forming membrane associations, and assembling the membrane skeleton(3) . This network of proteins was first defined in the red blood cell; however, many of the proteins present in the red cell membrane skeleton have closely related isoforms expressed in non-erythroid cells. The spectrin-actin lattice has thus far been visualized only in erythrocytes, and the organization of spectrin in different cells has yet to be defined.
Adducin (adducere: to bring together) was first identified as a
calmodulin-binding protein associated with the red cell membrane
skeleton(4) . Adducin binds to spectrin-actin complexes, is
able to recruit a second spectrin molecule to this ternary
complex(5) , and bundles actin filaments at high
concentrations(6, 7) . Association of adducin with
spectrin-actin complexes in in vitro assays has been
interpreted as occurring through an ordered pathway with the following
steps: spectrin and actin bind, adducin associates with the
spectrin-actin complex, and then an additional spectrin molecule
associates with the adducin-spectrin-actin ternary
complex(5, 8) . This protein is a substrate for
protein kinases A and C(9, 10, 11) , and
consists of two subunits, (molecular mass = 81 kDa) and
(molecular mass = 80 kDa), which are 49% identical and 66%
similar in amino acid sequence(12) . Adducin isoforms are
expressed in many tissues including brain, kidney, lung, liver, and
lens based on immunoblots and Northern blot
analysis(4, 12) . Adducin has been functionally
characterized from bovine brain as well as erythrocytes (9) .
In addition, several alternatively spliced isoforms have been described
for both the
(13) and
(12, 14, 15, 16) adducin genes.
In vitro activities of adducin suggest a function in
constructing the spectrin-actin lattice. The pattern of expression of
adducin is also consistent with a role in assembly of spectrin-actin
complexes. Adducin is expressed early in erythroid differentiation at
the proerythroblast stage and is present at the time when the spectrin
skeleton is assembled(17) . Adducin is localized at cell-cell
contacts with spectrin and actin in cultured epithelial cells (18) and no longer localizes to the plasma membrane of
epithelial cells when -spectrin is disrupted(19) . An
adducin-like protein is associated with the hts phenotype of
defective oocyte development in Drosophila(20) . The hts gene product is localized to ring canals, which are
abundant in actin filaments and represent a specialized region of
cell-cell contact. It has been speculated that mutant hts could affect ring canal formation either by disrupting actin
filament binding directly, or indirectly by interfering with spectrin
organization(21) . Adducin thus may be involved with actin and
spectrin in forming specialized contact sites in organisms as diverse
as Drosophila and mammals.
Adducin subunits are comprised
of structurally distinct domains: an NH-terminal 40 kDa
globular head domain, an 8 kDa neck domain, and a COOH-terminal domain
that has been predicted to be an extended
tail(12, 22) . COOH-terminal regions of adducin
subunits have not been isolated from native adducin due to their
protease sensitivity. These tail regions are difficult to visualize by
electron microscopy and have been referred to as ``fairy
tails'' due to their elusive properties. This report characterizes
COOH-terminal domains expressed in bacteria, presents evidence for an
extended configuration, and demonstrates that the tail domains of
adducin are responsible for association with spectrin-actin complexes.
In addition, domains involved in intersubunit contact between
-
and
-subunits in an adducin tetramer are defined, and a model for
organization of subunits in adducin tetramers is proposed.
Figure 1:
Ethylene glycol
bis(succinimidylsuccinate) (EGS) cross-linking of adducin to a
tetramer. I-Labeled red cell adducin (16 µg/ml, 1.3
10
cpm/µg) was incubated for 15 min at 2 °C
with 1 mg/ml ethylene glycol bis(succinimidylsuccinate) in a buffer
containing 10 mM sodium phosphate, 1 mM sodium azide,
1 mM DTT, 1 mM NaEDTA, and 0.05% Tween 20, pH 7.4.
The reaction was quenched with addition of glycine (10 mM final concentration), and samples were analyzed by SDS-PAGE
followed by autoradiography. Molecular weights were estimated using
human erythrocyte spectrin (M
= 260,000)
cross-linked to a dimer (M
= 480,000). Lane 1, uncross-linked adducin; lanes 2-5,
adducin + cross-linker in 0.1, 0.25, 0.5, and 1 M NaCl; lanes 6-8, adducin + cross-linker in 1, 2, and 4 M urea.
To assess the oligomeric
state of adducin, physical properties of the covalently linked tetramer
were determined in conjunction with those of the native uncross-linked
molecule (Table1). The Stokes radius for both species was
estimated by gel filtration. The native adducin peak is broad, giving
an average R of 6.9 nm. The cross-linked
adducin tetramer peak on the Superose 6 column is much sharper, giving
a Stokes radius of 8.0 nm and falls within the leading edge of the
uncross-linked adducin profile (Fig.2). These data suggest that
native adducin contains a mixture of dimers and tetramers either in a
static state or involved in a dimer-tetramer equilibrium. The nearly
quantitative formation of tetramer by cross-linking implies that during
the time of the cross-linking reaction (15-30 min) essentially
all dimers associate to form tetramers. Attempts were made to isolate a
cross-linked dimer form of adducin but were not successful.
Figure 2: Superose 6 gel filtration profiles of native adducin and cross-linked adducin. The Stokes radii of native adducin and cross-linked adducin were estimated by gel filtration on a Superose 6 column equilibrated in 10 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 1 mM NaEDTA, 0.05% Tween 20, pH 7.4. The Superose 6 column was calibrated with protein standards as discussed under ``Experimental Procedures.'' 0.5-ml fractions were collected for the native adducin, cross-linked adducin, and protein standard runs.
Molecular masses of cross-linked and native adducin were calculated to be 232 and 211 kDa, respectively, based on the Stokes radii from gel filtration and sedimentation coefficients determined by rate zonal sedimentation on sucrose gradients (Table1). The apparent molecular mass of cross-linked adducin is significantly lower than expected for a tetramer of 322 kDa and suggests an incorrect value for either the sedimentation coefficient or the Stokes radius. The most likely source of error is Stokes radius determined by gel filtration, with lower than actual values resulting from interaction of adducin with the gel matrix, or from anomalous behavior observed with other asymmetric proteins(37) . The tail domain of adducin is the source of aberrant behavior in SDS-electrophoresis as well as gel filtration (see below).
Figure 3:
Domain organization of adducin subunits. A, cartoon depicting the domains of an adducin subunit.
+++ represents the highly basic MARCKS-like region at
the COOH terminus of both subunits. B, Kyte-Doolittle
hydrophobicity plots of - and
-adducin. The hydrophilic tail
region of
-adducin encompasses residues 430-737, and the
-adducin tail contains residues 409-726. C,
Coomassie Blue gel of adducin domains. Lane 1, erythrocyte
membrane ghosts; lane 2, erythrocyte adducin; lane 3,
48-kDa core-neck fragment; lane 4, 40-kDa core fragment; lane 5, recombinant
-tail; lane 6, recombinant
-tail.
Circular dichroism indicated that COOH-terminal
tail regions of - and
-adducin are unstructured, with no
evidence for
-helix or
-sheet based on signals at 208 and 222
nm, and a negative peak at 200 nm which is indicative of a random coil (Fig.4B). Spectra of the COOH-terminal domains are in
contrast to those of intact erythrocyte adducin and the
NH
-terminal core region of adducin which contain
approximately 10-15%
-helix as well as
-sheet secondary
structure (Fig.4A). A thermal melt was performed on
both the adducin core and the
-adducin tail construct at 222 nm (Fig.5, A and B). Loss of secondary structure
in the NH
-terminal core can be seen at a midpoint of 59
°C with decreasing ellipticity at 222 nm. The
-adducin tail
appears to be heat stable, and the spectrum at 222 nm does not change
over the temperature range of 25-71 °C.
Figure 4:
Circular dichroism spectra of intact
adducin and adducin domains. Adducin tail domains are unstructured,
indicated by the negative peak at 200 nm. The intact and core adducin
are folded and contain an estimated 15 and 10% -helical secondary
structure. The percent
-helicity for the intact adducin and the
core fragment was estimated from measured ellipticities at 222 nm using
the algorithm of Chen and Yang (31) (see ``Experimental
Procedures''). A, intact red cell adducin and tryptic
core fragment (for preparation see ``Experimental
Procedures'') (40 µg/ml). B, recombinant
- and
-adducin tails (40 µg/ml).
Figure 5:
The COOH-terminal adducin tails are
extended, unstructured molecules and are heat-stable. The red cell
NH-terminal adducin core loses secondary structure
cooperatively at 222 nm at 59 °C, whereas the ellipticity at 222 nm
does not change over the temperature range for
-adducin tail.
Thermal melts were monitored at 222 nm by circular dichroism. A, erythrocyte adducin core. B,
-adducin
tail.
Circular dichroism
data support the idea of an unstructured COOH terminus, and the
protease sensitivity of this domain implies an extended, non-globular
structure. Molecular masses of 55 and 48 kDa calculated from the Stokes
radii and sedimentation coefficients indicate that - and
-adducin tails (34 kDa based on amino acid sequence) are either a
mixture of monomers and dimers, or that the Stokes radius has been
underestimated by gel filtration (see above) and the domains actually
are dimers with a true molecular mass of 68 kDa (Table1). The
frictional ratio for the tail constructs is estimated at about 1.3
using a molecular mass of 55 kDa, which was lower than expected.
However a corrected molecular mass of 68 kDa yields a frictional ratio
of 1.6, which is closer to the value anticipated from an extended
conformation. The calculated molecular mass of 232 kDa for cross-linked
adducin also is smaller than expected for a 320-kDa tetramer (see
above). The globular head domain, in contrast to native adducin and
COOH-terminal tail domains, behaves as expected on gel filtration and
SDS-electrophoresis (22) (Table1).
Figure 6:
Evidence of NH-terminal core
domain contacts. Limited V8 proteolysis of a cross-linked adducin
tetramer gives a stable 160-kDa fragment. A, Coomassie Blue
gel: lane 1, uncross-linked adducin; lane 2,
cross-linked adducin tetramer; lane 3, 10-min V8 digestion (3
µg/ml V8 protease); lane 4, 20-min V8 digestion; lane
5, 30-min V8 digestion. B, Western blot with an affinity
purified polyclonal antibody against the COOH-terminal tail of red cell
adducin: lane 6, cross-linked adducin tetramer; lane
7, 10-min V8 digestion; lane 8, 20-min V8 digestion; lane 9, 30-min V8 digestion. Electrophoretic transfer of
protein to the nitrocellulose filter was confirmed by Ponceau S
staining.
Radiolabeled
recombinant - and
-adducin tail constructs were used as
ligands to evaluate possible interactions with adducin and
NH
- and COOH-terminal domains immobilized on nitrocellulose
paper (Fig.7, panel A).
- and
-adducin tail
domains associated with tails of both subunits, as well as the intact
erythrocyte adducin protein (panels B and D). This
binding is specific, as evidenced by the displacement of binding when
1.3 µM unlabeled
- or
-adducin tail is included
in the incubation (panels C and E). Individual
subunit tails were able to form
-
,
-
, and
-
contacts. Neither the
- nor
-tails bound to the
NH
-terminal core.
Figure 7:
Association of - and
-adducin
tail domains. Erythrocyte adducin (lane 1),
NH
-terminal 40-kDa tryptic fragment (lane 2),
recombinant
-adducin tail (lane 3), and recombinant
-adducin tail (lane 4) were electrophoresed on SDS-PAGE
and were either stained with Coomassie Blue (panel A) or
transferred to nitrocellulose paper. The nitrocellulose paper was
blocked with 40 mg/ml bovine serum albumin in a buffer containing 10
mM sodium phosphate, 100 mM NaCl, 0.5% Tween 20, 0.2%
Triton X-100, 1 mM NaEDTA, and 1 mM DTT, pH 7.4. B,
I-labeled
-adducin tail (19,000
cpm/pmol) was added to 16 nM and incubated with moderate
shaking overnight at 4 °C. C, 1.3 µM cold
-adducin tail was added in conjunction with the
I-labeled ligand to assess nonspecific binding. D,
I-labeled
-adducin tail (57,000
cpm/pmol) was added to 16 nM and incubated under the same
conditions described for panel B. E, 1.3 µM cold
-adducin tail was added in addition to the labeled
-adducin tail.
Figure 8:
Recombinant - and
-adducin tails
bind to spectrin-actin complexes. A constant amount (20 nM) of
either
I-labeled
-adducin tail (5540 cpm/pmol) or
I-labeled
-adducin tail (52,000 cpm/pmol) was
separately incubated with spectrin (80 nM), polymerized rabbit
muscle actin (3 µM), and increasing amounts of unlabeled
- or
-adducin tail protein in a 75-µl volume. The
reaction buffer contained 50 mM KCl, 30 mM HEPES, 2
mM MgCl
, 1 mM NaEGTA, 10% sucrose, 0.05%
Tween 20, 1 mM dithiothreitol, and 1 mM ATP, pH 7.3.
After incubation, 70 µl of each reaction was layered onto a
150-µl sucrose barrier (20% sucrose in assay buffer) and was
centrifuged at 25,000 revolutions/min for 1 h at 4 °C in a Beckman
42.2Ti rotor. The supernatants and pellets were separated, and the
specific activity for each data point was recalculated to determine
spectrin-dependent picomoles adducin tail bound/nmol actin.
- and
-adducin tail binding to actin alone (0.42 pmol
-adducin
tail/nmol actin, 0.59 pmol
-adducin tail/nmol actin) has been
subtracted out from reported values.
Adducin has been seen to promote the binding of
spectrin to F-actin (5, Fig.9A). Fig. 9B shows that both the - and
-adducin tails can also
stimulate binding of spectrin to actin. The double-reciprocal plot for
this experiment estimated a K
of 100
nM for the red cell adducin; the adducin tails displayed a
2-3-fold lower affinity than the native adducin in this assay (K
= 270 nM). The
capacity of the native adducin-dependent binding of spectrin to actin
was similar to that of the recombinant adducin tail constructs (Fig.9C). The NH
-terminal 48-kDa core-neck
and 40-kDa core domain fragments did not show any activity over
background (spectrin binding to actin alone) in this assay (data not
shown). The extreme COOH terminus of both the
- and
-adducin
subunits contains a highly polybasic region of 22 amino acids that has
41% identity to the MARCKS protein(12, 38) . The
MARCKS protein was also analyzed using this assay but did not show any
spectrin-dependent binding to F-actin (data not shown). The polybasic
region at the extreme COOH terminus of the
- and
-adducin
subunits, which is similar to MARCKS, therefore is not sufficient to
bind spectrin-actin complexes.
Figure 9:
-Adducin and
-adducin tails
promote association of spectrin with actin in a saturable manner. A and B, cosedimentation assay with a constant amount (50
nM) of
I-labeled spectrin, F-actin (2.4
µM), and red cell adducin
, and
-adducin tails.
Buffer and incubation conditions were the same as reported in Fig.8. Binding data are averages of triplicate determinations.
Spectrin-actin binding in the absence of adducin (0.74 pmol
spectrin/nmol actin) has not been subtracted out from the reported
data. C, double-reciprocal plot for the adducin-dependent
binding of spectrin to actin data shown in panels A and B. Spectrin-actin binding in the absence of adducin (see
above) was subtracted from the saturation binding curve values and
plotted as reciprocal data. The lowest concentration point is not
shown. Red cell adducin stimulates binding of spectrin to actin with an
estimated K
of 100 nM, whereas
the adducin tails display a lower affinity than the native adducin,
estimated at about 270 nM. The capacities of the red cell
adducin and the adducin tails are similar.
This report characterizes the adducin molecule in terms of
oligomeric structure, folding of domains, and definition of domains
involved in contact with spectrin-actin complexes. Experiments with
cross-linked and native adducin indicate that adducin in solution is a
mixture of heterodimers and tetramers. Circular dichroism spectroscopy
of COOH-terminal domains of - and
-adducin expressed in
bacteria provides direct evidence for an unstructured random coil
configuration for the COOH-terminal region.
Some predictions for a
model of the adducin molecule can be made based upon the cross-linking,
physical properties, proteolysis, and blot binding data (Fig.10). Four amino-terminal head domains contact one another,
eliminating a model where two antiparallel dimers come together, to
form a tetramer. Exact orientation of the tails, whether two or four
-
,
-
, or
-
interactions, cannot be
assessed at the level of these experiments. The flexible, extended
tails would provide asymmetry to the molecule; intramolecular tail
contacts may also occur. Presumably these extended, protease-sensitive
tail regions are stabilized by their binding to spectrin and actin
filaments. The site on adducin for binding to spectrin-actin complexes
was identified as the COOH-terminal tail of both the
- and
-adducin subunits based on activities of expressed domains and
lack of activity of the NH
-terminal head domain.
Figure 10:
Possible orientation of adducin subunits
in the tetramer. This model for the adducin molecule shows a possible
equilibrium between adducin dimers and asymmetric tetramers of globular
cores with extended associated tails based upon cross-linking (Fig.6) and blot binding (Fig. 7) experiments. The
arrangement of - and
-adducin subunits in tetramers is not
known at this time. No evidence of any contacts between the tail and
the head domains of adducin was noted; however, the possibility of
tails looping backwards and binding to the neck domain cannot be
excluded. The COOH-terminal tail regions contain the site for
spectrin-actin binding and phosphorylation sites. The COOH terminus of
-adducin has also been shown to interact with calmodulin in a
Ca
manner.
Adducin
had previously been characterized as an /
heterodimer of 200
kDa, based upon migration on SDS-PAGE and physical
properties(4) . Properties of cross-linked adducin tetramer and
of COOH-terminal domains determined in this study provide evidence that
the tail domains have anomolous behavior on gel filtration and
SDS-polyacrylamide electrophoresis leading to underestimates of the
Stokes radius of the intact molecule and overestimates of monomer
molecular mass. An additional complicating factor is that adducin
exists in solution as a mixture of dimers and tetramers. Adducin
tetramers were inferred by previous electron microscopy studies, in
which the diameter of adducin and NH
-terminal
protease-resistant domains were identical and similar to predicted
dimensions of a 160-kDa globular protein(12) . The question of
whether adducin engages in a dimer-tetramer equilibrium or exists as a
static mixture of dimers and tetramers needs to be addressed in future
experiments. Adducin binds Ca
/calmodulin and accepts
phosphate from protein kinases A and C. Ca
/calmodulin
does not affect the profile of native adducin on gel filtration (data
not shown). However, the effects of phosphorylation on the oligomeric
state of adducin have yet to be tested.
Although adducin in
erythrocytes contains - and
-subunits in a 1:1 ratio, only
-adducin has been detected in keratinocytes (18) and
fibroblasts(39) . It is pertinent in this regard that the site
for adducin binding to spectrin-actin complexes has been mapped to the
COOH-terminal region of both
- and
-subunits, and that both
- and
-COOH-terminal domains can self-associate (Fig.7). Individual subunits should therefore be capable of
interacting with spectrin-actin complexes and at least of forming
homodimers. It will be of interest to determine if assembly of adducin
subunits to tetramers requires both
- and
-subunits and to
evaluate activities of
-adducin in the absence of
-adducin.
The COOH-terminal domain of adducin, although unstructured, is an
essential portion of the molecule. Activities in addition to
spectrin-actin binding, recruitment of additional spectrin molecules to
F-actin, and self-association include the site for
Ca/calmodulin binding on
-adducin(40) ,
and a binding site for stomatin (band 7.2b)(41) . The
COOH-terminal domain also is likely to contribute to functional
diversity among adducin isoforms, since alternatively spliced variants
have conserved NH
-terminal sequences and variable COOH
termini(13, 14, 15, 16) . The
NH
-terminal head domain, in contrast, is the most conserved
region of adducin but has no known role other than its role in
stabilizing the adducin tetramer. Determining the function(s) of the
NH
-terminal domain will be an interesting challenge for the
future.