The spectrin skeleton: from red cells to brain

The spectrin skeleton: from red cells to brain.

V Bennett, S Lambert

J Clin Invest.

1991;

87(5)

:1483-1489.

https://doi.org/10.1172/JCI115157

.

Research Article

Perspectives

The Spectrin Skeleton:

From Red Cells

to

Brain

Vann Bennett and StephenLambert

The HowardHughes MedicalInstituteand theDepartmentofBiochemistry, Duke University Medical Center, Durham, North Carolina27710

Introduction

Adevelopment from work with the plasma membrane of hu-manerythrocytes over the past two decades has been discovery

ofthespectrin-basedmembraneskeleton, elucidation of its

or-ganization, and isolationandcloning ofthemajor constituent

proteins. The membrane skeleton of

erythrocytes

has already hadimplications for hematologists in that defects of proteins involvedinthe membrane skeleton have been found to result in hereditary hemolytic anemias. The spectrin skeleton also has a much broader relevance with components highly

con-servedfrom Drosophilato man that are expressed in many cell types.Physiological functionsof spectrin skeletons in different

cells are likelyto be diverse but to involve the basic role of

providingorderfor integral membrane proteins within the

planeofthe lipid bilayerandofcouplingmembraneproteinsto componentsof thecytoskeleton. The focusofthis review will be on the current status of nonerythroid spectrinsand

anky-rins, whichareproteinsthat couplecertain integral membrane

proteinstospectrin.New developments with mutant mice will bedescribed suggestingthatthesamegenes forcertain erythro-cyte proteins are expressed in brain and can play a role in

neurological disease.

Overview

of

the

Membrane Skeleton

of

Human

Erythrocytes

Thespectrinskeleton can beviewedas a system of interacting

proteins coordinated intoanintegratedstructure. The skeleton thusis intermediateincomplexity betweenamultisubunit en-zyme and an organelle such as the endoplasmic reticulum.

High resolution electron microscopy of the stretched mem-braneskeleton hasprovided striking images ofa regular

lattice-likeorganizationwith five to sixrod-shaped spectrin molecules attached toshort actinfilaments 30-50nminlength toforma sheetof fiveandsix sidedpolygons (1, 2)(Fig. 1).

Spectrin,themajorstructuralcomponent of this network,

isa flexible rod-shaped molecule comprised of two subunits

aligned side-to-side toform heterodimers and head-to-head

intotetramers(Fig.2). Spectrin requires additional proteins to

formamembrane skeleton.Twoclassesof proteininteractions have beenidentifiedas essential forassembly of spectrin tetra-mersintoamembrane-associated network:

Linkage ofspectrin to the membrane. Spectrin molecules

Receivedforpublication

JODecember

1990andin revisedform 3 Febru-ary1991.

have twodistinct sitesof interaction withintegralmembrane

proteins thatarelikelyto occursimultaneously.Amajor

mem-braneattachment isprovided by

high-affinity

association ofthe betasubunit ofspectrinwith ankyrinat a site located in the

midregion ofspectrintetramers.

Ankyrin

isa

peripheral

mem-braneprotein which,in turn, is associated with thecytoplasmic domain of the anion exchanger.Another association of

spec-trin withthemembrane is mediated by protein4.1 which is

locatedatthe endsofspectrinmolecules andrecognizes

mem-branesitesthat may includeglycophorinC and the anion

ex-changer.

Association

of multiple

spectrin moleculeswith actin to

form

a two-dimensionalmeshwork. Spectrin molecules are

cross-linked at their ends by association with actin. Several

accessory

proteins

are foundatthesespectrin-actin junctions including protein 4.1 andprotein4.9. Otherproteinsarealso

candidatesto

participate

in

spectrin-actin

interactions

includ-ingadducin (3),

tropomyosin

(4), anda

tropomyosin-binding

protein named tropomodulin (5).

Spectrin

andthe

erythrocyte

membrane have been the

subject

of several recentreviews

(6-10).

Defects or

deficiency

in erythrocytemembrane skeletal

proteinsresultinabnormally

fragile

red cells and mildto severe

hereditary hemolytic anemias inhumansand mice. These

stud-iesestablish theprinciplethatthemembrane skeleton is

essen-tial fornormalsurvival of

erythrocytes

inthe circulation and thatdefectsinstructural

proteins

canbe the basis for disease

(reviewed in reference 11).

The Spectrin

Family

Spectrin

(also referredto as

fodrin)

includesagroupof

plasma

membrane-associatedproteinsthat are presentinmost

verte-bratetissues

(12)

(Table I).

Spectrins

have also been character-ized in nonvertebrates

including Drosophila

(13,

14)

and echinoderms (15)and must have evolvedbefore the

divergence

of arthropods.

Spectrins

havethe

following

consensus

proper-ties: (a)Morphology ofanextended, flexible molecule -

200-260 nm inlength comprised oftwodistinct extended

rod-shaped subunits ofmol wt

225,000-430,000.

The

subunits,

termed

alpha

andbeta,are

aligned laterally

inan

antiparallel

arrangement toform heterodimersandhead-head into tetra-mers 200nmin

length

in thecaseof themostcommonform

ofspectrin.

(b) Ability

toassociate withandcross-link actin

filaments. (c)A

calmodulin-binding

site located inthe midre-gion ofthealpha subunit ofmost

spectrins.

Calmodulin-bind-ing sitesare

missing,

however,in the caseof alpha

spectrins

of

mammalian erythrocytes and

Drosophila. (d)

An

ankyrin-binding site locatedonthe betasubunitofmost

spectrins

at a

site inthe

midregion

ofthetetramer

(16, 17).

Alphaand betasubunits of the

spectrin

family

are

homolo-gous to each otherandare

primarily comprised

of

multiple

J.Clin. Invest.

©TheAmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/91/05/1483/07 $2.00

Figure 1.Schematic model of the organization of the spectrin skeleton of human erythrocytes.

versions ofa 106-amino acid repeating sequence(18).Closely relatedversions ofthe 106-amino acid repeatingsequence of

spectrin subunitsare presentinproteins such as alpha-actinin

(19)anddystrophin(20) suggesting that theseproteins share a commonevolutionary originandforma newlydefined

super-family of proteins. Structural models have been proposed for thefolding of 106-residue repeats ofspectrin into a series of shortalpha-helicalsegmentscomprised ofeitherthree (18) or

four (21) alpha helices. The proposed series ofalpha-helical

units is consistent with the amount ofalphahelix predicted

from circular dichroism spectra, and would account for the reduced length andincreased flexibilityof spectrin compared

withothercoiled-coilalpha-helical proteins.

Mammals express two types ofalpha subunit which are

products ofdistinctgenes: atissue-invariantalpha subunit

lo-catedonhumanchromosome 9which isexpressedinall

mam-maliantissuesexceptformature erythrocytes (22), and the

erythrocyte alpha subunitlocated on human chromosome 1

(23). Birdsandpresumablyotheranimalshave asingle alpha

subunit expressedinerythrocytes aswell asothertissues. The betasubunits of spectrincontain most of the

recogni-tionsites of spectrin for other proteins including ankyrin( 17,

24),protein4.1(25),actin (26),as well as thesitefor

ankyrin-independent associationof spectrin with membranes (27). The

beta subunits also arethe majorsource ofdiversityamong

spectrins, withagrowing list of variants with specialized

func-tions. The beta subunit familycurrentlyincludesfive isoforms that arelikely to be encoded by distinctgenes: (a)

BetaG,

a

generally distributed tissue invariantpolypeptide that binds

preferentiallyto brain ankyrinas opposed to erythrocyte

an-kyrin.(b)BetaR, first characterized in erythrocytes that also is

expressed asalternatively spliced forms in brainand skeletal muscle (28). BetaR associates preferentially with erythrocyte

ankyrin

compared tobrain ankyrin.(c)

_Betarw,

a specialized

subunit associated with terminalwebinthe apicaldomain of

intestinal epithelialcells andwhich is foundinbirds but not in mammals(29).

BetaTw

may lack an ankyrin recognition site

based on in vitro assays(8). (d)

BetaNm,

abeta-type subunit

identifiedatneuromuscularjunctionsbased oncross-reactivity

withantibodies(30). The beta-related subunit at neuromuscu-lar junctions may be an exception to the general rule that beta subunits are associated with alpha subunits, because no alpha subunit could be detected by antibodies (30). (e) BetaH, a

430,000-D polypeptide initially discovered in Drosophila (31)

that forms tetramers with the alpha subunit that are 260 nm in length.

Severalexampleshave been noted where two beta subunits areexpressed in same cell but are localized in specialized do-mains. Avian intestinal epithelial cells have

betaTw

in their api-cal domains, whereas

betaG

is confined tothe basolateral

do-mains (29). Purkinjecells in the cerebellum that havebetaRin their cell bodies and beta0 in axons (32, 33). Expression of a

specialized beta subunit is turnedonduring differentiationof myoblasts(34),andislikelytobesubjecttointeresting

tissue-specificanddevelopmentalcontrols.

The completesequencehasbeendeterminedof cDNAs

en-codingthealphasubunits ofspectrinfrom chicken brain(35), human erythrocytes (36), Drosophila (14), and human lung

(37). The alpha subunitsoftissue spectrins have a very high

degreeof conservation of at least 90%identitybetween

verte-brate species and 63% identity between Drosophila and chicken. The human erythrocyte alpha subunit, in contrast, has 50-60%

identity

with the

general

human

alpha

subunit,

and currently represents the most divergent member of the

alpha spectrinfamily.

Themidregion ofallspectrin alpha subunitsincludesa se-quence that hashomologywith the SH3portionof the

regula-tory domains of severalsrc-tyrosine kinases and a domain of the gamma isoform ofphospholipase C (35). Other proteins that have asimilarmotif includeayeastactin-binding protein

andmyosin 1 ofDyctiostelium (38).All of theproteins

identi-fiedsofarwith aSH3motifhavethefeature ofassociation with the membranecytoskeleton (38). Intriguing possibilities

sug-gested bythe sequencehomologybetweenspectrinand other structuralproteinswith theregulatorydomains oftyrosine ki-nases arethat these enzymes areregulated by interactions with structuralproteinsorthat the SH3 motif isresponsiblefor

tar-geting

a

variety

of

proteins

tothe

vicinity

of the

plasma

mem-branethrough association witha commonclassofmolecules.

Sequence information for the betaspectrins includes the

completesequence of humanerythrocyte betaR (28),and

par-tial sequences for

Drosophila

betaG

(13) and betaH (31). BetaR containsthreedistinctregions:anNH2-terminaldomain

(resi-dues1-272)thatisacandidatetocontain the

actin-binding

site

(26),adomain comprisingthemajorportion ofbeta

spectrin

composed of17consecutive 106 residuerepeats,anda COOH-terminal domain of 52 residues that is

responsible

for head-head association withinthe NH2-terminus of the alpha

sub-unit.The

ankyrin-binding

siteis

likely

tobelocatedin repeat 15, whichhas a stretchofsequence that

diverges

from the

adducin protein 4.1

actin _ankyrin

al p ha C N

calmodulin SH3 domain

TableI. The Spectrin Family

Subunits* Proteins Tissue

Alpha,

Alpha.,

beta,

Generally distributed Alphas Alphab,beta$ Erythrocytes

Beta,

Alpha.,

beta,

Brain, muscle

Beta,

Alpha.,

betatv, Avian intestine

Betart

Alpha?, betanm Neuromuscular junction

Beta..

Betanm

Betah§

* Arbitrary nomenclature forthe purposeof this review.

$Encoded by thesame gene asbetarbutalternatively splicedwith an extensionattheCOOH-terminal end (28).

1_{Recently discovered in Drosophila (31); localization in vertebrates}

not known.

106-residuerepeatmotif(28). Drosophila beta spectrins both

have high homology within the NH2-terminal actin-binding

domain, with 77% sequence identity(13). The actin-binding

domain ofbetaspectrinalsoisconserved between members of the spectrin/alpha actinin/dystrophin gene family as well as otheractin-bindingproteinsthatassociatelaterally with actin

filaments includingDictyostelium gelation-factor (39)and nonmusclefilamin (40).

Ankyrins

Ankyrins, like spectrins,are a family of proteins that are

asso-ciatedwiththeplasmamembranesofmany typesofcells.

An-kyrins have propertiesthat suggest a role as adaptors between

certainintegral membrane proteinsand thespectrinskeleton.

Erythrocyte ankyrin,thefirstmemberofthis family to be

char-acterized,providesahigh-affinity linkagebetween spectrin and thecytoplasmic domain oftheanionexchanger(41).Ankyrin

isalargeprotein of 206-kDthatcontains three independently-folded domains:(a) anNH2-terminal 89-kDdomain that binds totheanionexchanger (42);(b)a62-kDdomain(apparent mol wt 72,000 on SDS gels) that binds to spectrin (43); and (c) a

COOH-terminal 55-kD regulatory region comprised ofatleast twodomainsthat modulates activityofthebindingdomains

(44) (Fig. 3). The complete sequence of human erythrocyte

ankyrinhas been deducedfromanalysis ofcDNA which

en-codesa

protein

of 1,881 amino acids from a mRNAof7 kb

(45, 46).

Astriking feature ofthe 89-kD domain of ankyrin is the presenceof22 repeatscontaining 33-residuesthat occur in tan-dem. The repeatscontain 15highlyconserved and 18 variable

residues. Related 33-residue motifsare present in a number of

apparently unrelated proteins of broadphylogenetic

distribu-tion (see45for references):(a)cytoplasmic domains of mem-braneproteins involvedin celldifferentiationincluding Lin 12 andGlp-1 of

C. elegans,

Notch protein of

Drosophila

and

XotchofXenopus (47);(b)cytosolic proteins involved in

cell-cycle regulation such as

SWl6

and

SWl4

of S. cerevisiae and CDC10ofS.

pombe

where the33-residuemotif was first noted

(48); (c)the precursor toNf-kappaB, aubiquitous transcrip-tion factor (49,50). Thefunctional basisfor these homologous sequences is not clear, although a shared interaction with a commonclass ofmoleculesisareasonable

guess.

The89-kD domain is globular and has a CD spectrum

con-sistent with 30% alpha helix (42). These

physical properties

providesome boundaryconditions for predictions of the orga-nization and folding ofthe 33-amino acid repeatingsequences. Thevalueof 30%alphahelix impliesasinglehelixof10

resi-dues per repeat ifit isassumed that each repeat is folded ina

quasiequivalentmanner. Theglobular shapeof the 89-kD do-main suggests that the repeatsfold intoasphereandnotinto an

extended rod as is the case ofmany proteins with multiple

repeated sequences. Itisof interest that another form of

an-kyrin from brainalso has22repeatsof 33 amino acids

(Otto,

E.,M.Kunimoto,and V. Bennett,

manuscript

in

preparation).

Themaximum numberof

ankyrin

repeats thus may be 22 if the repeats arepacked intoasphere.

Immunoreactive forms of ankyrinhavebeendetected

asso-ciated withthemembranes ofanumber oftissues inaddition

to erythrocytes by radioimmunoassay, immunoblots ofSDS

gels, and immunofluorescence (41,

51).

Anisoform of

ankyrin

has beenpurified from brain whichhas

properties

incommon

with erythrocyteankyrin,althoughit is theproduct ofadistinct

gene(17) (see below). Brain and

erythrocyte ankyrin

share

phys-ical

properties

(asymmetric

monomersof molwt

200,000),

have asimilar domain structure, associate with thebeta sub-unit of

spectrin

at a site close to the

midregion

of

spectrin

tetramers, andboth

proteins

bindtothe

cytoplasmic

domain of

theerythrocyte anion exchanger. Thetwo

ankyrins

also share the propertyofbindingtotubulinvia the 89-kD domain.

Recent workindicatesthaterythrocyteandbrain

ankyrins

areprototypesoftwofamilies of

ankyrin

that have

major

dif-ferences incellular

expression

andlocalization

(52, 53).

Anky-rinR

forms orrestricted

ankyrins

reactbetter with antibodies against RBC

ankyrin,

are

expressed

inalimited number of cells inbrain and kidney, andhave a

highly polarized

distribution withinthese cells.

_AnkyrinR

in thenervoussystemis

expressed

primarily inneurons,andin

kidney

ispresent in

high

concen-trations in distal tubule cellsandintercalated cells of the

col-lectingduct(52, 54).

AnkyrinR

formsarelocalizedat

special-ized celldomains suchasthe node of Ranvier

(53),

postsynap-tic membrane of the neuromuscular

junction (55),

and

basolateral surfaces of

epithelial

cells

(51, 54).

AnkyrinR

also is

MISSING IN 2.2

89K 62K

33-aa repeats _Regulatory Domain

Regulatory

domain

tubulin

present in nerve cellbodies, at the initial segment ofaxons,

dendrites,and inunmyelinatedaxons(53).

Several ion channels colocalize withankyrinRinspecialized membranedomainsandinteract with erythrocyte ankyrin in

invitroassays:theanionexchangerofkidneycollecting ducts (56), the alpha1isoform oftheNa/K ATPaseofkidney (54, 57, 58), andthevoltage-dependent sodium channel of brain (59).

A current workinghypothesisisthatankyrin plays arolein

eitherinitialtargeting of theseionchannelstospecializedareas

ofthecell or inmaintainingthem once the membranedomains

have assembled.

Membersofthe

ankyrinB

group cross-react betterwith anti-bodies against the major form of ankyrin in brain, and are expressed in mostcellsofbrainandkidney.

AnkyrinB

ispresent

in kidneyinglomeruli, proximalanddistal tubules,andloops

ofHenle (52).

AnkyrinB

inbrain ispresentinglialaswellas

neuronalcells, and does notexhibitahigh concentrationatthe

nodesof Ranvier. Neither form of ankyrin ispresent in

inter-nodal regions of myelinated axons, at least as detected with available antibodies,eventhoughthese areasof the membrane

contain spectrin. One candidate fora membrane attachment

site for

ankyrinB

forms include a broadly distributed

mem-braneglycoprotein, termed Pgp- 1, gp-85, or CD44antigen (60), which islikely to participate in intercellular adhesion.

Anotherankyrin-binding proteintermed AGP-200 has been

detected in brain (61). Recent experiments in ourlaboratory using

ankyrinB-affinity

columns suggest that multiple

mem-braneproteins recognize this form of ankyrin(62).

Differences in cellular localization of ankyrinsmay be due tovariationsinrelative affinities fortargetproteins. Functional differences betweenthe

_ankyrinR

and

ankyrinB

isoforms have been demonstrated usinginvitroassaysemploying erythrocyte andbrain ankyrinasprototypesof these families.Each typeof ankyrin binds preferentiallyto adistincttypeof spectrin: brain ankyrin better withthegeneral form ofspectrinand

erythro-cyteankyrinbetterwith erythrocyte spectrin(17,63).The

speci-ficity

of ankyrins for spectrin isoforms is

likely

tobeimportant indifferential targeting of ankyrinsatleast inbrain whereboth

erythroidand generalspectrinarecoexpressed inthe same cells butlocalizedindifferent domains of certainneurons(32, 33).

Membranebinding sites for ankyrinsalso aredistinctinbrain

and

kidney

(52). The

specificity

of membrane sites for ankyrins in kidneywasnotabsolute,butreflected differences in relative affinities. Further evidence forcommon features in

binding

siteswasthatthecytoplasmic domain of the erythrocyte anion exchangerdisplaced membrane binding of both ankyrins.

cDNA

encoding

ankyrinB

from human brainhasrecently

been cloned and sequenced (Otto, E., M. Kunimoto, and V.

Bennett,manuscriptinpreparation). Brain ankyrin isencoded

byagenelocatedonchromosome 4(Ottoetal.,manuscriptin

preparation), whereas the gene for erythrocyte ankyrin is lo-cated onchromosome8(46).The openreading frame of brain ankyrin cDNA encodes aprotein of202kD, which iscloseto

thesize of erythrocyte ankyrin.Twomajor regions of high

ho-mologytoerythrocyteankyrinarepresent in the sequenceof brain ankyrin: oneinvolves the33-residue repeatswhich are present in 22 tandemcopies inboth proteins, andthe other area of

homology

is locatedwithin the spectrin-binding

do-mains. Conservation within therepeatdomains ofthese

anky-rins includes preservation of the number ofrepeats, andthe feature that the fourthrepeat inboth

proteins

is the onlyrepeat

todeviate fromthe33-residue

periodicity.

Moreover,each

re-peat in brainankyrin is morecloselyrelatedtothe correspond-ingrepeat inerythrocyteankyrin, stronglysuggesting that these sequencesaroseby duplicationofanancestoral gene that also

contained22copiesof this motif.

Twoareasof almost complete divergence between the two

ankyrinsare atthe connection between the 33-residue repeat

domain and thespectrin-binding domain, and in the regula-tory domains. These areas of sequence differences are candi-date sites to explain the functional differences between brain andRBCankyrins.

Howmanydifferent genes encode ankyrins? The informa-tion to answer this quesinforma-tion still is incomplete,althougha min-imum number may be five distinctankyringenes. Northern blotsandimmunoblots with antibody raisedagainst recombi-nantproteinsindicate that the twoankyrinsfromkidneyare distinct from both brain and RBCankyrin,and that liver has a

uniqueform ofankyrinnotpresent inbrain, RBCs,orkidney

(Otto et al., manuscript in preparation). Moreover, in brain tissue,

ankyrinR

atthe node of Ranvier may be encodedbya

different gene than

ankyrinR

in neuronal cell bodies because NB/NBmice aremissingthemajorform of

ankyrinR

but still express anankyrin at their nodes of Ranvier(64).

A rational nomenclaturetodescribethese different

anky-rins has not beenformulated,andprobablywillrequire addi-tional information. Simplyusingthetissueorigintodesignate

ankyrins is not sufficient. For example, the samegene that encodes erythrocyte ankyrinalso isresponsible foraform of ankyrin in the cerebellum and forebrain(64,65).

Functional

Diversity

ofAnkyrin

Dueto

Alternative

Splicing

of

mRNA

Erythrocyte ankyrin has aregulatoryregioncomprisingseveral

domains whicharelocatedatthecarboxy terminalend of the

polypeptide. Oneof these domains,locatednearthecarboxy

terminus, is cleaved bycalpainand results inanankyrinwith reduced bindingto theanion exchangerinerythrocyte

mem-branes(44). Anotherregulatory domain is located

NH2-termi-nal to the calpain-sensitive domain. Deletion of this domain

results in a lower molecularweight form of

ankyrin

presentin

humanerythrocyte membranesknown as

protein

2.2.Protein 2.2 resultsfrom differential processing of mRNA,because the

missing region, as identified by

antibodies,

lies

internally

within the sequence (45). Moreover,cDNAclones have been

isolatedthat contain anin-frame deletion of163 amino acids that include the portion of sequence

missing

in

protein

2.2 (45,46).

Thealternatively splicedprotein2.2isanactivated

ankyrin

with anincreasedaffinity for spectrinandincreased association

with the anionexchangerin erythrocyte membranes(44).

Pro-tein 2.2also expresses a

binding

site fora

major

classof

uniden-tifiedprotein sitesinkidney microsomes that donot

recognize

the larger form of ankyrin (52). The regulatory

region

thus definesspecificityinbindingtomembranesitesaswellas mod-ulatesaffinities.Theexplanation for increased activitiesof

pro-tein2.2 isnotknown. One

possibility

isaconformational

dif-ference between2.2 and intact

ankyrin.

Another alternative is

thatthedomain

missing

in 2.2

occupies

binding

sitesas a

pseu-dosubstrate, as occurswith regulatory domains ofseveral

pro-tein kinases.

Thephenomenonof alternative splicing of

ankyrin

mRNA

islikelytoinvolve regionsinadditiontothe

region

missing

in

family.Inthecaseoferythrocyte ankyrin, a highly basic stretch

of32residues(pl > 10) located at the COOH-terminus of the regulatory domain is also alternatively spliced (46). Another

candidate sitethat would have asignificant functional conse-quencewouldbe in theregioninterconnecting the 89-kD and

spectrin-bindingdomains. A potential example of splicing in

brain involves

ankyrinB

which exhibitstwo sizesof mRNA on Northern blots that encodes two ankyrins with quite different regulatorydomains (Otto et al., manuscript in preparation).

Mapping

the Binding Sites ofAnkyrin

How does ankyrin interact selectively with the anion ex-changer, Na/K ATPase, andvoltage-sensitivesodiumchannel aswell as other membrane proteins that remain to be

identi-fied?Theavailableevidence,based onexaminationofthe

bind-ing sites of ankyrin forthe anionexchanger and Na/K ATPase supportstheview that ankyrin-bindingactivity evolved

inde-pendentlyatleastinthe caseoftheseproteins(42). The anion exchangerbinds exclusivelyto the 89-kD domain (see below), whereas the Na/K ATPase binds only weakly to the 89-kD

domainand alsoassociates withthe spectrin-binding domain

(66). The Na/KATPasethus may require contacts with two andperhaps moredomains ofankyrin to form the high-affinity

complexobserved with intact ankyrin.

Theanion exchanger-binding siteof ankyrin iscompletely contained withinthe89-kDdomain ofhuman erythrocyte

an-kyrinandis retainedbyproteolytic fragments containing only

33-residuerepeats (42). The33-residuerepeats thus play a

ma-jor

roleinassociation ofankyrin withtheanionexchanger. The

issue ofwhetherthe repeats areequivalentwith respect to

bind-ing to theanion exchanger has been explored using defined

regions of humanerythrocyte andbrain ankyrins expressed in

bacteria (67). The conclusionwas that the repeats are not

inter-changeableand that the 44residues from722 to 765 are

essen-tial forhigh-affinity binding betweenerythrocyteankyrinand theanionexchanger.

Thefindingthatspecificity in associationbetweenankyrin andthe anionexchangeris providedby a rather smallregion of sequenceraises thequestion ofwhat is the function(s) of the

remaining 20 33-residue repeats of the 89-kD domain. One

possibility is that the other 33-amino acidrepeatsinteractwith

proteins distinct fromtheanion exchanger. The ability to inter-actwith

multiple

proteins isnotrequired in thecontextofthe

anucleate mammalian erythrocyte. However, the same gene

encoding erythrocyte ankyrin also isexpressed in brain (64,

65). The complex environment of the nervous system could provide a variety of potential ankyrin-binding proteins.

Spectrin and

Ankyrin

in

Specialized

Membrane Domains

Isoforms ofboth ankyrinandspectrinare segregated to special-ized regions ofcellmembranes, and may play a role in either

establishing ormaintaininglocal concentrations of integral

membraneproteins.Ankyrin associates and is colocalized with severalpolarizedmembraneproteins, includingtheanion

ex-changer in basolateral domains of kidney collectingducts(56),

thevoltage-dependentNachannel at nodesofRanvier ofnerve

(54)

andthe neuromuscular

junction

(55)and theNa/K ATP-aseof basolateral domains of kidney distaltubule cells(54, 57,

58). Examples

of

highly

polarized

distributions of spectrinare

theconcentration of

spectrin

atthe nodeof Ranvier (68),and in caps oflymphocytes in tissues (69). Spectrin also is a

signifi-cantcomponentofpostsynaptic densitiesof brain(70).A vari-antof the beta subunit of spectrin is associated with the

post-synapticmembraneofthe neuromuscularjunctionand is colo-calized withacetylcholinereceptors(30).

Theseobservationsestablishthatspectrin,eitherdirectlyor through ankyrin, has the potential for localization in special-izedregions ofcell membranes. Animportantissue is whether

spectrinandankyrin play a role in establishing membrane

do-mains. Arangeofpossibilities include participationin initial

targeting ofmembraneproteins, stabilizationofmembrane do-mainsafterthey haveformed,orhavingarole in both mainte-nanceandinitialassemblyofmembrane domains. A complex

containingtheNa/KATPase, ankyrin, andspectrinhas been

isolatedfromculturedepithelialcells that isdetergentsoluble and may represent a precursor to the mature,detergent insolu-ble assembly of these proteins on the plasma membrane (71).

Ankyrin and theNa/K ATPase thus arelikelytoassociate

be-fore the Na/K ATPasereaches the plasma membrane,

al-though it isnot knownwhether theseproteinsinteract in the

Golgiorendoplasmicreticulum. How theankyrin/ATPase complex is targetedtobasolateralmembranes remainsanopen

question. It seemslikely that information from the external

sideoftheplasmamembrane will be involved in thetargeting

event.

Clinical

Implications

The phenotype of anemia has established the importance of spectrin and its associated

proteins

in normalfunction of

eryth-rocytes. In view ofthe widespreadnature ofthese structural systems,it islikelythat thespectrinskeleton may be involved

in aspectrum ofcell membrane-related diseasesinother tis-sues.Giventhediversenatureofthesestructural

proteins,

aris-ing frombothmultiplegenes andalternativemRNAsplicing,a large numberoftissue-specific defectscould beforeseenthat would becompatible withsurvival.

Agood starting point inthesearch for such diseases would

be

proteins

suchasbetaR

spectrin

andankyrinRwhichare

en-codedby thesamegenes inerythrocytes, skeletal muscle,and some neurons in thecentral nervoussystem. The NB/NB strain ofmice, for example,lackerythrocyteankyrinand also aredeficientinthis form ofankyrinin thecerebellum,whereit isalmostcompletelymissing from thecellbodies of

Purkinje

and granule cells (64, 65). Otherankyrin genesstill are

ex-pressed including ankyrinatthe nodeofRanvier andthe

major

form of brain

ankyrin.

These animals

develop

atremor and other

signs

of cerebellar

dysfunction

(65). Moreover, the

Pur-kinje cellsgradually die until at age 6-9 mo the number is

reducedby 50% (65).Adefect inaspecific

ankyrin

gene thus

can leadto neuronal

degeneration

in these miceand

poten-tiallyin humans as well. Itisofinterestthat

neurological

symp-tomshave beenreported associated with

hereditary

spherocy-tosis (72-75), althoughthese cases may berelatively rare. In

view ofthepotential for multiple ankyrin-binding proteins withinaneuron, it islikelythatdefectsinankyrincanoccur

which do notaltertheactivityin erythrocytes but will affect

neuronalfunction.

Aprediction fromtheNB/NBmousemodel isthatdefects

in some membersofankyrinorspectrin familieswill result in

degenerative diseases of

long-lived

cellssuch as neuronsand musclecells. These

proteins

havemultiple isoformsthat may be abletocompensate intheshort term,butmaynotbeableto

diseases may becurrentlyviewedas anormalconsequence of

aging.

Ms.BrendaSampsonisgratefully acknowledgedforhelpinpreparing themanuscript.

Researchfromourlaboratorywassupportedinpartbygrants from

theNationalInstitutes of Health.

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