From the Department of Cell Biology and Biochemistry and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adducin is a protein associated with spectrin and
actin in membrane skeletons of erythrocytes and possibly other cells.
Adducin has activities in in vitro assays of association
with the sides of actin filaments, capping the fast growing ends of
actin filaments, and recruiting spectrin to actin filaments. This study
presents evidence that adducin exhibits a preference for the fast
growing ends of actin filaments for recruiting spectrin to actin and
for direct association with actin. -Adducin-(335-726) promoted
recruitment of spectrin to gelsolin-sensitive sites at fast growing
ends of actin filaments with half-maximal activity at 15 nM
and to gelsolin-insensitive sites with half-maximal activity at 75 nM.
-Adducin-(335-726) also exhibited a preference for
actin filament ends in direct binding assays; the half-maximal
concentration for binding of adducin to gelsolin-sensitive sites at
filament ends was 60 nM, and the Kd for
binding to lateral sites was 1.5 µM. The concentration of
-adducin-(335-726) of 60 nM required for half-maximal binding to filament ends is in the same range as the concentration of
150 nM required for half-maximal actin capping activity.
All interactions of adducin with actin require the myristoylated
alanine-rich protein kinase C substrate-related domain as well as a
newly defined oligomerization site localized in the neck domain of
adducin. Surprisingly, the head domain of adducin is not required for
spectrin-actin interactions, although it could play a role in forming
tetramers. The relative activities of adducin imply that an important
role of adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or
loss of actin subunits.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ubiquitous expression of spectrin and spectrin-associated proteins in most cells of metazoan organisms raises the issue of how spectrin-based structures are assembled and regulated. The erythrocyte membrane skeleton is the best characterized example of a spectrin-actin network, although spectrin also is associated with membranes at sites of cell-cell contact in epithelial cells, along axons in the nervous system, and at specialized sites in striated muscle (1). Spectrin in erythrocytes is organized into a polygonal network formed by 5-7 spectrin molecules linked to short actin filaments about 40 nm in length (2-4). Spectrin-actin junctions contain a group of proteins that presumably have functions in promoting spectrin-actin interactions, forming connections with the membrane, and regulation of actin filament length (1, 5).
Adducin is one of the proteins localized at spectrin-actin junctions
(6) and was originally purified based on calmodulin binding activity
(7). Adducin also is a substrate for protein kinase C and protein
kinase A (8-10). Adducin phosphorylated at the major protein kinase C
site is localized in dendritic spines and axonal growth cones of
cultured neurons, indicating adducin is a protein kinase C substrate
in vivo (11). Adducin is encoded by three closely related
genes termed ,
, and
adducins which are expressed in many
types of cells (7, 9, 12). Adducins all contain an N-terminal globular
head domain, a neck domain, and a protease-sensitive tail domain with a
C-terminal basic stretch of 22 amino acids with homology to the
MARCKS1 protein (9, 12, 13).
The MARCKS-related domain of adducin contains the major sites for both
protein kinase C phosphorylation and calmodulin binding (10). Adducin
is a mixture of heterodimers and heterotetramers in solution, with
tetramers formed by four head domains in contact with one another to
form a globular core, and interacting tail domains extending away from
the core (14). Adducin oligomers in erythrocytes comprise
/
subunits and in other cells include
/
as well as
/
combinations of subunits (7, 9).
The patterns of expression and cellular localization of adducin are consistent with a role in interaction with spectrin in erythrocytes as well as other cells. Adducin is localized at spectrin-actin junctions in mature erythrocytes (6) and is expressed early in erythropoiesis at a time when the spectrin-actin network is forming (15). Adducin and spectrin are both concentrated at sites of cell-cell contact in epithelial tissues and at dendritic spines and axon growth cones of cultured neurons (11, 16). Expression of a dominant-negative form of spectrin that inhibits assembly of spectrin tetramers results in loss of epithelial polarity and disassociation of adducin from the plasma membrane (17). Hts, an adducin-related protein in Drosophila, is required for the formation of ring canals, an actin-rich structure, and is co-localized with spectrin in developing germline cells (18, 19). Spectrin and adducin also have been observed associated with a dynactin complex which contains an actin-related protein (20).
Adducin activities determined in in vitro assays include recruiting spectrin to actin filaments (21, 22), bundling actin filaments (23, 24), and capping the fast growing ends of actin filaments (25). Each of these individual adducin activities may reflect different aspects of adducin function in living cells. In this study we have quantitated the relative affinities of adducin for actin capping, direct binding to actin, as well as recruiting spectrin to the sides and fast growing ends of actin filaments. The conclusion of these experiments is that adducin preferentially associates with and recruits spectrin to the fast growing ends of actin filaments. We also demonstrate that adducin interactions with spectrin and actin require the MARCKS-related domain of adducin as well as a newly defined oligomerization site localized in the neck domain of adducin.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
EZ-LinkTM Biotin-BMCC, NeutrAvidin, and dithiobis(succinimidyl propionate) were from Pierce. Electrophoresis reagents and the Bradford protein assay solution were from Bio-Rad. Epoxide-modified microspheres (0.35 µm diameter; MMA/GMA copolymer) were from Bangs Laboratories. Standard microcentrifuge tubes (400 µl) were from Eppendorf. 125I-Bolton-Hunter reagent was from ICN Radiochemicals. Pepstatin A, dithiothreitol, phenylmethylsulfonyl fluoride, and benzamidine were from Sigma. Uranyl acetate was from Electron Microscopy Sciences. Copper grids (400 mesh) were from Mason & Morton Ltd. 4-(2-Aminoethyl)-benzenesulfonyl fluoride was from Boehringer Mannheim. S-Sepharose resin and Mono Q columns were obtained from Amersham Pharmacia Biotech.
Methods
Procedures-- Protein concentration was determined by the procedure of Bradford (26). SDS-polyacrylamide gel electrophoresis was performed using 0.2% SDS with buffers of Fairbanks et al. (27) on 1.5-mm thick 3.5-17% exponential gradient gels. Quantitation of proteins by the pyridine dye elution method was basically as described (28). Briefly, the bands corresponding to the targeted proteins were cut out from the Coomassie Blue-stained SDS-polyacrylamide gel and the dyes were extracted in 25% pyridine. The relative amounts of protein were measured by reading the absorbance at 595 nm of dye/pyridine solution.
Protein Purification--
Actin was purified from acetone powder
of rabbit skeletal muscle (29) with a modification that actin monomer
was isolated by gel filtration chromatography on a Superose 12 column
before the final polymerization step. Bovine brain spectrin was
isolated following high salt extraction from brain membranes (30). Red blood cell adducin was purified from the low salt extract of human erythrocyte membranes as described previously (14). Purified human
plasma gelsolin was purchased from Cytoskeleton. Purified recombinant
human plasma gelsolin expressed from Escherichia coli was a
generous gift from Dr. Paul A. Janmey, Harvard Medical School, Boston.
-Adducin constructs were generated using full-length human
-adducin cDNA (12) as a polymerase chain reaction template and
were subcloned into a Studier plasmid with a T7 promoter (31). Each
adducin polypeptide has three additional N-terminal amino acids
(Met-Ala-Ser). Procedures for subcloning and bacterial expression of
the constructs were as described (32). The expressed proteins were
purified as follows. Bacterial pellets were resuspended in 100 ml of 50 mM sodium phosphate (pH 7.4), 1 mM EGTA, 25%
sucrose, 10 mM MgCl2, 0.04 mg/ml DNase I, and
protease inhibitors including 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 mM
benzamidine, 0.01 mg/ml pepstatin A. The suspension was mixed with 200 ml of extraction buffer containing 200 mM NaCl, 20 mM sodium phosphate (pH 7.4), 2 mM EDTA, 1%
Triton X-100, 1 mM DTT, and protease inhibitors as
mentioned above. Subsequently the lysate was forced through a 20-gauge
needle once and centrifuged for 20 min at 5,000 × g. The recombinant proteins were in the Triton-soluble fraction, which was
then loaded onto a 15-ml S-Sepharose column. The protein was eluted
with 0.5 M NaCl in 10 mM sodium phosphate (pH
7.4), 1 mM EDTA, 1 mM NaN3, 0.05%
Tween 20, 1 mM DTT. Optimal column fractions were pooled
and dialyzed against the same elution buffer but with 50 mM
NaCl. The protein was loaded onto a 10-ml Mono Q high pressure liquid
chromatography column and eluted by a linear 0.05-0.5 M
NaCl gradient. Generally, a 2-liter culture can result in 5-10 mg of
protein with over 90% purity.
Preparation of Immobilized Actin-- Purified F-actin was incubated with EZ-LinkTM Biotin-BMCC for 5 h with a 5:1 molar ratio of biotin versus actin. Then the biotinylated F-actin was depolymerized by dialysis for 48 h against G buffer containing 2 mM Tris (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT. Avidin beads were prepared as follows. NeutrAvidin was coupled to epoxide-beads for 48 h in a coupling buffer containing 10 mM HEPES (pH 7.0), 1 M NaCl, 1 mM sodium EGTA, 1 mM NaN3. The reaction was quenched by a buffer containing 100 mM Tris (pH 8.0), 1 M NaCl, 1 mM EGTA, 1 mM NaN3 and then the beads were washed twice with coupling buffer. Subsequently the biotinylated and depolymerized actin was incubated with avidin beads at 4 °C for 5 h. The amount of actin bound to the beads (usually 4-9 µg per µl of packed beads) was monitored by measuring free actin before and after the coupling using Bradford assays. The immobilized actin were washed twice by a buffer containing 2 mM Tris (pH 8.0), 50 mM KCl, 2 mM MgCl2, 1 mM ATP, 0.5 mM DTT. In the final step, beads coupled actin and un-biotinylated actin monomer (with a ratio of 1:9) were co-polymerized in 2 mM Tris (pH 8.0), 50 mM KCl, 2 mM MgCl2, 1 mM ATP, 0.5 mM dithiothreitol.
Actin Co-sedimentation Assay-- The co-sedimentation assay using free actin filaments was described previously (14, 22). The co-sedimentation assay using immobilized actin was performed as follows. 125I-Labeled ligand (adducin or spectrin) was incubated with immobilized actin (0.4 µM actin) in a 60-µl volume for 1 h at 4 °C in a buffer containing 30 mM HEPES (pH 7.0), 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 10% sucrose, 0.05% Tween 20, 2 mg/ml bovine serum albumin, 0.5 mM ATP, 0.2 mM DTT, 0.5 mM NaN3. The incubated mixtures were then layered onto 200 µl of 20% sucrose dissolved in the same incubation buffer in 400-µl microcentrifuge tubes and centrifuged for 10 min at 4,000 × g. Supernatants and pellets were separated by freezing the tubes on dry ice and cutting the tips of the tubes. The radioactivity of both the supernatant and the pellets was analyzed with a gamma counter. The amount of actin in pellets was determined in parallel experiments under the same conditions.
Actin Polymerization Assay-- Pyrene-labeled actin was prepared as described (33). G buffer (2 mM Tris (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT) was used in all assays. The assay quantitated inhibition of actin polymerization at barbed ends utilizing the method of Pollard (34) in which rapid polymerization was initiated using F-actin nuclei. Briefly, 4 µM G-actin was mixed with adducin, and actin polymerization was initiated by adding 0.25 volume of 1.25 µM F-actin nuclei in 10 mM Tris (pH 8.0), 250 mM KCl, 5 mM MgCl2. Actin polymerization was followed by monitoring the increased pyrene fluorescence of labeled F-actin (excitation at 365 nm and emission of 407 nm) during 30-210 s after initiation of the polymerization using a spectrofluorimeter. The sample temperature was maintained at a constant 25 °C for all experiments using a circulating water bath.
Electron Microscopy-- Immobilized actin or free actin in a buffer containing 30 mM HEPES (pH 7.0), 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.05% Tween 20, 0.5 mM ATP, 0.5 mM NaN3 was applied to a 400-mesh copper grid coated with carbon film for 30 s and washed by 10-20 drops of 1% uranyl acetate. The grid was then air-dried for 5 min and examined by a Philips EM 301 electron microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Use of Immobilized Actin to Study Adducin/Actin/Spectrin Interactions-- Adducin/actin/spectrin interactions were previously monitored by a binding assay using actin filaments as the sedimentation matrices (14, 21, 22, 32). Recent studies showed that adducin caps the fast growing ends of actin filaments (25). In order to evaluate directly interactions of adducin with actin filament ends, we developed a technique for measuring binding to short actin filaments with relatively more ends than in the conventional assay. Biotinylated actin was coupled to avidin beads (referred to as "immobilized actin"). Beads were selected to be (a) impermeable to proteins to minimize the background due to trapping, (b) small (0.35 µm) to maximize surface/volume ratio, and (c) of appropriate density (1.1 g/ml) to sediment through a sucrose barrier. Immobilized actin sediments at 4,000 × g for 10 min, and free actin requires high centrifugation forces (up to 80,000 × g for 60 min) to pellet. Compared with the conventional assay using free actin, the immobilized actin assay can reduce the background that results from sedimentation of ligands independent of actin.
The filament lengths of immobilized actin were compared with those of uncoupled actin filaments (referred to as "free actin" in this article). Fields of negatively stained immobilized actin contain much fewer extended actin filaments than a sample of free actin, even though both preparations contain equal concentrations of actin (Fig. 1A). It suggests that immobilized actin has more short filaments which are coupled to the beads. Moreover, the number of actin filament ends are determined quantitatively by using gelsolin to cap the fast growing ends of actin filaments (35). Actin/gelsolin co-sedimentation was performed in a buffer at pH 7.0 and calcium-free to minimize actin filament severing activity of gelsolin (36, 37). Gelsolin does not bind to beads alone but binds to immobilized actin with a ratio of approximately 1 gelsolin/15 actin monomers (Fig. 1C, lanes 3 and 4). In contrast, gelsolin binds to free actin with a ratio below 1 gelsolin/250 actin subunits (Fig. 1C, lanes 1 and 2), suggesting much fewer filaments ends available in free actin samples.
|
|
Adducin Polypeptides Containing Only Neck and Tail Domains Are
Functionally Equivalent to Native Adducin in Spectrin Recruiting and
Actin Capping Activities--
Contributions of individual domains to
adducin function were evaluated using recombinant polypeptides
expressed in bacteria (Fig. 3). We
focused on -adducin as an initial step and have confirmed the major
findings with
-adducin. The domain boundaries of adducin subunits
were inferred from patterns of polypeptides produced by limited
proteolytic digestion of erythrocyte and brain adducin (13, 22). Mild
chymotryptic digestion of adducin yields an N-terminal fragment of 48 kDa (residues 1-436), whereas tryptic digestion produces an N-terminal
fragment of 39 kDa (residues 1-354), implying a chymotrypsin-resistant
domain of 80 residues (355-435) which is referred to as the neck
domain. Twenty additional N-terminal residues were added to constructs
encompassing the neck domain to compensate for possible errors in
estimates of molecular weight by SDS-electrophoresis (Fig. 3). The tail
construct (residues 409-726) designed previously by Hughes and Bennett
(14) lacks most of the neck domain. Thus
-adducin residues 335-436 and 437-726 are designated to be the neck and the tail domains, respectively, and were expressed in bacteria with a three amino acid
addition at the N terminus but otherwise no additional residues (see
"Methods"). The head domain (residues 1-354) was not included due
to the limited solubility when expressed in bacteria (data not
shown).
|
|
|
The MARCKS-related Domain of Adducin Is Necessary but Not
Sufficient for Interactions with Spectrin and
Actin--
-Adducin-(335-694), which lacks the MARCKS-related
domain, almost completely lacked activity in promoting association of spectrin with actin filaments (Fig. 4).
-Adducin-(335-694) also exhibited an 80% reduction in the extent of actin capping activity (Fig. 5) and in actin binding activity (Fig.
6A). The residual activities
of
-adducin-(335-694) in actin capping and actin binding assays are
in the range of nonspecific effects. The MARCKS-related domain is not
sufficient for activities, however, since a construct,
-adducin
residues 437-726, with the MARCKS-related domain and the rest of the
tail, but lacking the neck domain, also exhibited minimal spectrin
recruiting activity, actin capping activity, and actin binding activity
(Figs. 4-6). These experiments are the first evidence that the
MARCKS-related region is necessary (but not sufficient) for adducin
functions. A role for this domain is consistent with the previous
findings that calmodulin, which binds to the MARCKS-related domain
(10), inhibits spectrin recruitment (21) and actin capping (25).
Moreover, phosphorylation of the MARCKS-related domain by protein
kinase C also inhibits actin capping and spectrin recruiting activities
(11). The MARCKS-related domain is highly conserved in all adducin
subunits, so these results suggest a general principle that this domain
is essential for adducin/actin interactions.
|
The Neck Domain of Adducin Is Required for Interactions with
Spectrin and Actin and Self-associates to Form Oligomers--
Deletion
of the neck domain from -adducin-(335-726) resulted in the loss of
almost all the spectrin recruiting and 80% of actin capping activities
(Figs. 4 and 5). The neck domain by itself had no detectable spectrin
recruiting (Fig. 4) or actin capping activity (Fig. 5). These findings
suggest that the neck domain is necessary but not sufficient in itself
for adducin interactions with spectrin and actin. Adducin is known to
associate into dimers and tetramers (7, 14, 22), suggesting the
possibility that one role of the neck domain could be in formation of
oligomers. A possible correlation between adducin domains and
oligomerization was evaluated by chemical cross-linking experiments.
Fig. 7A shows that
polypeptides containing the neck plus tail and neck alone were
cross-linked into oligomers. The major form of oligomer is dimer, but
trimer, tetramer, and higher oligomers also are evident. In contrast,
the construct lacking the neck region,
-adducin-(437-726), was not
cross-linked and remained a monomer. The cross-linking reaction
reflects specific protein-protein interactions because the majority of
the cross-linked products were abolished in the presence of 6 M urea. It is noteworthy that native erythrocyte adducin
forms tetramers as the major cross-linked products (Fig. 7A), suggesting that additional oligomerization sites must
exist which further connect
- and
-adducin dimers into
hetero-oligomers (Fig. 7A).
|
Fast Growing Ends of Actin Filaments Participate in Both Actin
Binding and Spectrin Recruiting Activities of Adducin--
The
relationship between the actin binding and actin capping activities of
adducin was evaluated by comparing the structural requirements and
relative affinities for these activities. The structural requirements
for actin binding and actin capping activities of adducin are similar,
i.e. both the neck domain and the MARCKS-related region are
needed for full activity (Figs. 5 and 6). However, actin binding and
actin capping require different adducin concentrations for half-maximal
activation and exhibit a major difference in the number of binding
sites. -Adducin-(335-726) associates with actin filaments with a
Kd of 1.5 µM and a capacity of about 1 adducin monomer/actin monomer estimated from double-reciprocal plots
(Fig. 6). The capacity of actin filaments for
-adducin-(335-726) cannot be explained by fast growing ends of actin filaments, which can
only account for approximately 1/16 of total actin, based on Fig.
1B. Moreover, the actin binding affinity
(Kd = 1.5 µM) is 10-fold lower than
capping activity (Kcap = 150 nM, Fig. 5). These results indicate that the major class of sites for
adducin in the actin binding assay are low affinity 1:1 complexes with
subunits exposed on the sides of actin filaments and are in agreement
with previous observations of association of adducin with the sides of
actin filaments (23, 24).
|
|
|
Spectrin Is Preferentially Recruited by Adducin to
Gelsolin-sensitive Sites on Actin Filaments--
The spectrin
recruiting activity of -adducin-(335-726) was measured as a
function of adducin concentration in the presence and absence of
gelsolin (Fig. 10). The
gelsolin-sensitive component of spectrin recruiting exhibits a
half-maximal activation at 15 nM
-adducin-(335-726),
whereas the gelsolin-insensitive spectrin recruiting was half-maximal
at 75 nM
-adducin-(335-726). The extent of
gelsolin-sensitive spectrin recruitment reached a maximum of 1 site per
1,000 actin monomers at 50 nM adducin. The extent of
spectrin recruitment depends on the spectrin concentration, which was
only 1 nM in this assay and does not represent the actual capacity for spectrin. Nevertheless, the fact that gelsolin-sensitive spectrin recruitment reached a maximum whereas the gelsolin-insensitive recruitment continued to increase indicates a limited number of gelsolin-sensitive sites that were considerably less than the number of
gelsolin-insensitive sites. The ratio of
gelsolin-sensitive/gelsolin-insensitive spectrin recruitment varied
from about 5:1 or higher, at
-adducin-(335-726) concentrations
below 25 nM, to 1:1 at
-adducin-(335-726)
concentrations of 200 nM. These results indicate that at
low adducin concentrations spectrin is preferentially recruited to
gelsolin-sensitive sites at the fast growing ends of actin
filaments.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study presents evidence that adducin exhibits a preference
for the fast growing ends of actin filaments for recruitment of
spectrin to actin and for direct association with actin.
-Adducin-(335-726) promoted recruitment of spectrin to
gelsolin-sensitive sites at fast growing ends of actin filaments with a
half-maximal activity of 15 nM and to gelsolin-insensitive
sites with half-maximal activity of 75 nM.
-Adducin-(335-726) also exhibited a preference for actin filament
ends in direct binding assays; the half-maximal concentration for
binding to filament ends was 60 nM, and the Kd for total actin binding was 1.5 µM.
The concentration of
-adducin-(335-726) of 60 nM
required for half-maximal binding to filament ends is in the same range
as the concentration of 150 nM required for half-maximal
actin capping activity. All interactions of adducin with actin require
the MARCKS-related domain as well as an oligomerization site defined
for the first time in the neck domain. Surprisingly, the head domain of
adducin is not required for spectrin-actin interactions, although it
could play a role in forming tetramers. This information is summarized
in a schematic model, which is drawn with the assumption that the tail
domains lie along the groove of actin filaments (Fig.
11). A previous model depicted that
adducin capped F-actin with the tails toward the slow growing ends of
actin (25). Because of the new information from this study, which
include the necessity of MARCKS-related domain and the sufficiency of
neck-tail for the actin capping activity of adducin, we propose the
model in which adducin tails oriented toward fast growing ends of
F-actin and MARCKS-related domains mediate direct contact with actin
ends (Fig. 11).
|
The relative activities of adducin imply that an important role of adducin in cells is to form a complex with the fast growing ends of actin filaments that recruits spectrin and prevents addition or loss of actin subunits. Adducin is the first example of an actin-capping protein that recruits other proteins to actin filament ends and could represent a new class of assembly factor with the function of integrating actin into other cell structures. The number of spectrin molecules recruited per adducin is not yet known. However, a possibility consistent with available data is that one spectrin tetramer is stabilized for each adducin dimer, and two spectrin molecules are associated with adducin tetramers. In this case, spectrin tetramers, which contain two actin binding domains, would form a one-dimensional chain which could be the structural precursor to the two-dimensional network of the mature erythrocyte membrane skeleton (2-4). Additional spectrin molecules associated with lateral sites along actin filaments may be stabilized by protein 4.1, which is present in approximately one copy per spectrin dimer. Actin filaments in erythrocyte membrane skeletons are likely to contain tropomyosin, associated along the filament groove (40), and tropomodulin, which binds tropomyosin and caps the slow growing ends of actin filaments (41), as well as dematin (42). It will be of interest to evaluate association of adducin with actin complexed with these accessory proteins. A prediction is that adducin will be excluded from sites on actin filaments coated with tropomyosin and will be confined to filament ends (5).
The fast growing ends of actin filaments of erythrocyte ghosts or isolated spectrin-actin complexes include a population capable of supporting addition of actin monomers and therefore are not capped (2, 43). Fowler and colleagues (44) have recently found that actin filament ends in intact erythrocytes are inaccessible to the actin-capping protein CapZ, which is exclusively localized in the cytosol. However, extraction of adducin in low ionic strength buffers lacking magnesium exposed binding sites for CapZ. These findings considered together with the actin capping activity of adducin are consistent with capping of actin filaments in unlysed erythrocytes by adducin (44).
A technical innovation that facilitated this study was an actin sedimentation assay using immobilized actin. The increase in signal is interpreted as resulting from the decrease in filament length and an accompanying increase in the number of actin filament ends (Figs. 1 and 2). Possible reasons for the presence of short filaments are as follows: 1) immobilized actin forms nuclei for actin polymerization with each immobilized actin subunit participating in one filament, and 2) shear force induces fragmentation of beads-coupled long actin filaments. Relatively few direct measurements of protein interactions with actin filament ends have been reported in the literature, with the majority of studies focusing on inhibition of actin polymerization. The assay described here could be useful for direct analysis of interactions of other actin-capping proteins with filament ends.
An oligomerization domain of adducin was mapped to the neck region,
encompassing residues 335-436 of -adducin (Fig. 7). Within the
stretch of 335-436, residues 360-386 are highly conserved among human
- and
-adducin subunits (12), rat
-adducin (9), and
Drosophila Hts (18). This 360-386 region has the highest probability in full-length
-adducin to form an amphiphilic
-helix with hydrophobic residues distributed along one side (not shown). Amphiphilic
-helices can form coiled-coil structures and mediate protein oligomerization, and the region 360-386 is one candidate for
contacts in oligomers. In support of a role for the region 360-386,
mutation of methionine 369 to proline reduced oligomerization activity
of adducin neck-tail (not shown). The fact that the neck domain forms
tetramers as well as dimers indicates that subunit contacts in addition
to a single coiled-coil interaction also are likely to be involved in
oligomerization. The precisely controlled 1:1 ratio of
/
subunits
of erythrocyte adducin (7) could be a consequence of the large excess
of
- over
-subunits (12) and lack of stability of
-homodimers.
Alternatively, some mechanism could exist that specifies assembly of
adducin heterodimers. The head domain of adducin may be responsible for
additional subunit contacts in heterodimers or tetramers. In support of
the idea of subunit contacts in addition to the neck domain,
cross-linking of adducin followed by proteolysis results in a 160-kDa
tetramer of 40-kDa monomers that lack neck domains (14).
The MARCKS-related domain is demonstrated in this paper to be necessary for interactions of adducin with spectrin and actin. The MARCKS-related domain of adducin also is the site of calmodulin binding and contains the site of phosphorylation by protein kinase C, which both regulate spectrin recruiting and actin capping activities of adducin (10, 11, 21, 25). The polybasic MARCKS-related domain of adducin is a candidate to provide a direct contact with actin and to participate in ionic interactions with negatively charged residues on actin. The idea of an electrostatic interaction between adducin and actin is supported by the finding that spectrin recruiting activity of adducin is salt-sensitive.2 The tropomyosin/actin interaction is mediated by the charged residues on the sides of actin filaments (45), suggesting the possibility that adducin could also bind to actin filaments in a similar manner and perhaps compete with tropomyosin. Possible residues involved in a complex with adducin at the fast growing ends of actin filaments include Asp-288 and Asp-286. These residues have been proposed to participate in salt bridges between actin monomers in actin filaments (46, 47). The increased stability of adducin complexes with the ends of actin filaments could result from participation of both lateral contacts and the contacts with residues such as Asp-288/Asp-286 at filament ends. The atomic structures of actin complexed with gelsolin segment 1 and with profilin implicate apolar residues of actin (48, 49). However, profilin also binds charged residues of actin (Asp-288, Glu-361) (49) which may be the targets of the MARCKS-related domain. These considerations suggest that steric inhibition rather than direct overlap of binding sites is likely to be a major factor in the gelsolin inhibition of adducin/actin binding (Fig. 8).
Adducin is unusual in its ability to associate with both the sides and ends of actin filaments. Tensin is another protein with multiple actin binding activities and caps F-actin, binds along the sides, and bundles the filaments (50). However, the actin binding and actin capping activities of tensin are located in different domains. The unique feature of adducin is that the same regions, residues 335-726, including both an oligomerization domain and the MARCKS-related domain, are required for actin capping, direct actin binding, and spectrin recruiting activities.
Polybasic domains similar to the MARCKS-related domain of adducin are
present in the MARCKS protein family and the NR1 subunit of the NMDA
(N-methyl-D-aspartate) receptor. The polybasic
domain of the MARCKS protein also is the target for both calmodulin
binding and phosphorylation and is directly involved in actin binding and cross-linking (51). The polybasic domain of the NMDA receptor is
the site for high affinity calmodulin binding and phosphorylation by
protein kinase C (52). The findings of this study suggest the
hypothesis that the NR1 subunit of the NMDA receptor also may interact
with actin and possibly promote either spectrin-actin or
-actinin-actin interactions (53).
Competition between gelsolin and adducin, utilized in this study as a probe for adducin interactions with the fast growing ends of actin filaments, may also occur in vivo considering the ubiquitous expression of adducin and gelsolin (9, 12, 54). A consequence of gelsolin occupation of actin filament ends instead of adducin would be displacement of spectrin and disassembly of spectrin-actin networks. A variety of pathways regulate actin capping activities, such as calcium dependence of gelsolin (35, 55), phosphoinositide inhibition for gelsolin and Cap Z (56, 57), and calcium/calmodulin inhibition and phosphorylation inhibition for adducin (11, 25). A recent study suggested that phosphorylation of adducin by Rho kinase enhances the adducin/actin interaction (58). These considerations suggest that adducin could contribute to a coordinated and elaborate regulation of actin-based structures.
![]() |
ACKNOWLEDGEMENTS |
---|
Dr. Harvey Sage is gratefully acknowledged for performing the sedimentation equilibrium experiments. Carmen Lucaveche is thanked for the expert guidance for electron microscope techniques. Dr. Paul Janmey is thanked for the purified recombinant gelsolin. Dr. Christine Hughes is acknowledged for the initial studies on the adducin neck-tail construct.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-684-3538;
Fax: 919-684-3590.
1 The abbreviations used are: MARCKS, myristoylated alanine-rich protein kinase C substrate; DSP, dithiobis(succinimidyl propionate); DTT, dithiothreitol; EZ-LinkTM Biotin-BMCC, 1-biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido] butane; NMDA, N-methyl-D-aspartate.
2 X. Li, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|