Proteins of the Spectrin- based Membrane Skeleton 
or leave the axon through ion channels. Localiza- 
tion of the voltage-dependent sodium channel at 
nodes of Ranvier is important for normal conduc- 
tion of nerve impulses. We hope to identify the 
gene encoding the form of ankyrin at the node of 
Ranvier and eventually to understand the role of 
nodal ankyrin in organization of this specialized 
membrane domain. These studies will have rele- 
vance to diseases of neurons such as multiple scle- 
rosis, where the myelin coating of axons is lost 
and sodium channels are no longer restricted to 
the nodes of Ranvier. 
Diversity of ank)'rins suggests that this family of 
proteins may interact with many membrane pro- 
teins. Characterized membrane proteins in brain 
that associate with ankyrin in in vitro assays in- 
clude the voltage-dependent sodium channel and 
sodium/potassium ATPase. We have used the 
membrane-binding domain of the major form of 
brain ankyrin to isolate ankyrin-binding proteins 
and have identified a family of ankyTin-binding 
proteins found in plasma membranes of neurons 
and glial cells. These ankyrin-binding proteins 
represent 0.3 percent of adult brain membrane 
protein and appear late in postnatal develop- 
ment. An important goal for future work will be 
to isolate the cDNAs encoding these proteins and 
determine their function in adult brain. 
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 have discov- 
ered that calcium, in concert with three differ- 
ent calcium-regulated proteins (calmodulin, a 
calcium-activated protease, and protein kinase 
C), inhibits the direct spectrin-membrane link- 
age but has no effect on spectrin-ankyrin interac- 
tions. These results suggest that the spectrin skel- 
eton includes both stable, ankyrin-mediated 
linkages and dynamic calcium-sensitive associa- 
tions that are subject to metabolic control. Iden- 
tification of the spectrin "receptor" is the first 
step in understanding the role of this type of 
spectrin-membrane interaction in cells. 
Ankyrin Structure 
Ankyrin contains three independently folded 
domains: one that interacts with certain mem- 
brane proteins, another that associates with spec- 
trin, and a third that regulates associations of the 
binding domains. Surprisingly, the membrane- 
binding domain of ankyrin includes an amino 
acid sequence that is homologous to regions of 
sequence in a group of apparently unrelated pro- 
teins from flies, yeast, and even viruses. We have 
recently discovered that this conserved portion 
of the ankyrin sequence is responsible for the in- 
teraction of ankyrin with at least one membrane 
protein. 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, with the expectation that this 
structure will help us understand interactions 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 and are in the process of 
determining a physical model for organization of 
domains and subunits in the adducin molecule. 
Future experiments will evaluate the role of ad- 
ducin and spectrin in formation of junctions be- 
tween cells. 
34 
