Proteins of the Spectrin- based Membrane Skeleton 
dance in the cerebellum and brain stem. Brain 
thus contains ankyrins encoded by different 
genes. The erythrocyte form of ankyrin is missing 
in a strain of mutant mice developed at the Jack- 
son Laboratory. The ankyrin-deficient mice expe- 
rience degeneration of Purkinje cells, a major 
type of neuron in the cerebellum, and develop a 
stagger and difficulty in walking. These mice re- 
tain ankyrin at the nodes of Ranvier, suggesting 
that ankyrin at the nodes is the product of yet 
another gene. The neurological problems of an- 
kyrin-deficient mice may have counterparts in 
humans w^ith slowly progressive diseases due to 
death of nerve cells. 
The diversity of ankyrins suggests that this fam- 
ily of proteins may interact with many membrane 
proteins in addition to the sodium channel. We 
have used the membrane-binding domain of the 
major form of brain ankyrin to isolate ankyrin- 
binding proteins and have identified at least 10 
new proteins. The consequence of linkage of 
these proteins to the membrane skeleton could 
be to localize them in specialized regions of the 
cell membrane appropriate for their function. 
Ankyrin-Independent Membrane 
Attachment Sites for Spectrin 
Brain spectrin can also associate directly with 
membrane proteins through an interaction that is 
independent of ankyrin. We recently discovered 
that calcium, in concert with three different cal- 
cium-regulated proteins (calmodulin, a calcium- 
activated protease, and protein kinase C) inhibits 
the direct spectrin-membrane linkage but has no 
effect on spectrin-ankyrin interactions. These re- 
sults suggest that the spectrin skeleton includes 
both stable, ankyrin-mediated linkages and dy- 
namic calcium-sensitive associations that are sub- 
ject to metabolic control. Identification of the 
spectrin "receptor" is the first step in under- 
standing the role of this type of spectrin-mem- 
brane interaction in cells. We are currently char- 
acterizing a protein that is a candidate for a role in 
linking spectrin to the membrane. 
Ankyrin Structure 
Ankyrin contains three independently folded 
domains: one domain interacts with certain mem- 
brane proteins, another associates with spectrin, 
and a third regulates associations of the binding 
domains. Surprisingly, the membrane-binding 
domain of ankyrin includes an amino acid se- 
quence that is homologous to regions of se- 
quence in a group of apparently unrelated pro- 
teins from flies, yeast, and even viruses. We 
recently discovered that this conserved portion 
of the ankyrin sequence is responsible for inter- 
action of ankyrin with at least one membrane pro- 
tein. Ankyrin thus contains a highly conserved 
and ancient structural motif that may have a gen- 
eral role in molecular recognition. We hope to 
determine the three-dimensional structure of this 
portion of ankyrin and expect that this struc- 
ture will increase understanding of the interac- 
tions of ankyrin and other proteins with related 
sequences. 
Adducin 
The protein adducin is a candidate to play a 
role in assembly of the spectrin skeleton in eryth- 
rocytes, brain, and certain epithelial tissues. We 
have found that adducin is localized at sites of 
cell-cell contact in epithelial tissues. Adducin 
and spectrin are colocalized at cell contact sites 
and may be arranged in a structure analogous to 
the spectrin network of erythrocytes. The associa- 
tion of adducin with cell-cell contact sites occurs 
before assembly of other types of specialized cell 
junctions such as desmosomes. 
Our working hypothesis is that adducin pro- 
motes assembly of a stable spectrin network at 
sites of cell-cell contact. A further hypothesis is 
that the spectrin network is an essential precon- 
dition for assembly of specialized cell junctions. 
Formation of appropriate cell-cell contacts and 
cell junctions is an essential event in embryogen- 
esis and is one of the processes that is disturbed in 
diseases such as cancer. We are excited by the 
possibility that adducin and spectrin may partici- 
pate in such a fundamental activity of cells. We 
have determined the complete protein sequence 
of both adducin subunits. Future experiments 
will evaluate the role of adducin and spectrin in 
formation of junctions between cells. 
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