MEMBRANE-CYTOSKELETON INTERACTIONS 
Vann Bennett, M.D., Ph.D., Investigator 
The long-term goals of this laboratory are to ad- 
vance understanding of the structural basis for 
long-range order in plasma membranes of mamma- 
lian cells. Research has focused on a system of pro- 
teins associated with plasma membranes of most 
animal cells, the spectrin-based membrane skele- 
ton. Spectrin and its associated proteins are candi- 
dates for a role in localization of integral membrane 
proteins at specialized regions of the plasma mem- 
brane and in assembly of cell-cell junctions. As an 
initial approach to the functions of spectrin in com- 
plex tissues, this laboratory is examining the associ- 
ation of spectrin with the plasma membrane. An- 
kyrin is a spectrin-binding protein that links the 
spectrin skeleton to membrane proteins. Ankyrin- 
binding proteins include the anion exchanger of 
erythrocytes and cells of kidney collecting ducts, 
the al isoform of the Na''^,K^ ATPase, and the volt- 
age-dependent sodium channel of brain. These an- 
kyrin-binding proteins have in common the feature 
of existing in specialized regions of the plasma 
membrane as an essential aspect of their function 
in tissues. The goal is to understand the molecular 
basis for the dual requirements of diversity and se- 
lectivity of protein interactions that allows ankyrin 
to associate with a number of different membrane 
proteins localized in distinct membrane domains. 
I. Mechanisms of Ankyrin Diversity. 
A. Ankyrin isoforms. The discovery of an associa- 
tion of ankyrin with the sodium channel in in vitro 
assays was unanticipated, since the sodium channel 
is highly localized to nodes of Ranvier and initial 
segments of axons, whereas ankyrin was believed 
to be distributed widely in brain. However, recent 
work indicates that ankyrin in brain and kidney is a 
family of closely related proteins that associate with 
spectrin but have different membrane binding sites 
and are targeted to distinct regions of the plasma 
membrane. Antibodies have been used to distin- 
guish two variants of ankyrin in the nervous system. 
Localization of these ankyrin isoforms in brain has 
revealed that 1) one isoform is expressed only in 
neurons, whereas the other is present in both neu- 
rons and glial cells; and 2) the neuronal isoform of 
ankyrin is highly concentrated at nodes of Ranvier 
and initial axonal segments, sites that contain the 
voltage-dependent sodium channel. A specific inter- 
action of this isoform of ankyrin with the sodium 
channel may play an important role in the mor- 
phogenesis and/or maintenance of the nodes of 
Ranvier. 
DNA encoding a human brain isoform of ankyrin 
has been isolated and partially sequenced. This iso- 
form of brain ankyrin has extensive homology with 
human erythrocyte ankyrin in the spectrin-binding 
domain and in a region that associates with the 
anion exchanger and perhaps other membrane 
proteins. Brain ankyrin is the product of a distinct 
gene from erythrocyte ankyrin, and it is likely that 
additional genes will encode the ankyrin isoforms 
of kidney and other tissues. Another ankyrin iso- 
form of human brain also has been cloned, al- 
though only limited sequence is available at this 
time. It will be important to determine the scope of 
the ankyrin family and to elucidate functional dif- 
ferences among family members. Dr. Bennett antici- 
pates from previous work that different ankyrins 
will have distinct membrane receptors and associ- 
ate with different isoforms of spectrin. 
B. Deletion of regulatory domains by alternative 
splicing of mRNA. Erythrocytes contain a form of 
ankyrin missing a regulatory domain but retaining 
binding sites for spectrin and the anion exchanger. 
The shortened version of ankyrin exhibits en- 
hanced binding to spectrin and to membrane sites. 
Dr. Bennett and his colleagues have discovered 
that the regulatory domain deleted from activated 
erythrocyte ankyrin is located internally within 
the protein sequence and thus results from alter- 
native splicing of mRNA. Alternative splicing of 
mRNA leading to variation within the regulatory do- 
main also is likely to occur in brain and provides an 
additional level of diversity within the ankyrin- 
family. 
II. Structural Basis for Association of Erythrocyte 
Ankyrin with Membrane Proteins. 
The binding sites involved in association between 
ankyrin and the anion exchanger have been ex- 
plored. The ankyrin-binding site of the anion ex- 
changer includes a region in the middle of the 
cytoplasmic domain but requires an extended se- 
quence of at least 100 amino acids for full expres- 
sion of activity. The anion exchanger-binding site of 
ankyrin has been localized to a 90,000-M^ amino- 
terminal fragment (90,000 domain) containing 22 
Continued 
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