identification of the functional site of erythrocyte protein 4.1 involved

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 hy The American Society of Biological Chemists, Inc.

Val. 261, NO. 7, Issue of March 5, pp. 3310-3315 1986 Printed in dI.S.A.

Identification of the Functional Site of Erythrocyte Protein 4.1 Involved in Spectrin-Actin Associations*

(Received for publication, July 26, 1985)

Isabel Correas, Thomas L. Leto, David W. Speicher, and Vincent T. MarchesiS From the Department ojPathology, Yale University School of Medicine, New Haven, Connecticut 06510

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a CAMP-dependent phosphorylated site.

The membrane skeleton of the mammalian erythrocyte is composed of a two-dimensional network of structural proteins which participate in a number of membrane-related phenom- ena. The major constituent, spectrin, is a long, rod-like molecule capable of multiple associations with other skeletal components. The formation of a two-dimensional lattice has been attributed to a combination of the self-assembly of spectrin into branched oligomers at the “head” region of the spectrin molecule (1) and the cross-linking of actin filaments with the “tail” region of spectrin ( 2 ) . Attachment of the spectrin-actin complex to the overlying membrane is mediated by two linking proteins, ankyrin and protein 4.1. Protein 4.1 greatly enhances the affinity of spectrin for actin filaments and is capable of forming a stable ternary complex in vitro (3, 4). The nature of this complex (5,6) and the effects of spectrin and protein 4.1 on the size and growth of actin filaments are currently subjects of intensive investigation in several labo- ratories (7-9). Protein 4.1 also enhances the binding of actin filaments to membrane vesicles (10) and is thought to serve as a link between the membrane skeleton and the cell’s exterior through specific interactions with transmembrane glycoproteins (11,12). The high affinity association of protein 4.1 with glycophorin appears to involve polyphosphoinositides as essential binding cofactors (13). Recently, we have shown that protein 4.1 also serves as a substrate for several protein kinases (14, 15).

Current interest in the red blood cell cytoskeleton as a model for membrane-cytoskeleton interactions has been greatly stimulated by the identification ofprotein 4.1 (16-19) and spectrin-like molecules (for a review, see Ref. 20) in a

*This research was supported by National Institutes of Health Grant GM-21714. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom all correspondence should be addressed.

variety of non-erythroid cells. An immunocross-reactive iso- form of protein 4.1 in brain has been identified as synapsin I (211, a multiply phosphorylated protein associated with syn- aptic vesicles (22). Isoforms of protein 4.1 in fibroblasts and endothelial cells appear to associate with actin filaments when analyzed by immunofluorescence (16, 23). Spectrin-like proteins also bind to F-actin (24-27), and in the case of brain spectrin, or fodrin, this binding is promoted by protein 4.1 (27). Taken together, these findings suggest that stabilization of spectrin-actin filament associations is a general role of the non-erythroid protein 4.1 isoforms as well.

In this study, we have identified the functional site of protein 4.1 which affects spectrin-actin filament associations. Using quantitative sedimentation assays, full activity in promoting spectrin-actin associations was demonstrated within an active fragment of 8,000 daltons generated by mild proteolytic cleavage of isolated protein 4.1.

EXPERIMENTAL PROCEDURES

Protein Purification-Protein 4.1 was purified from human eryth- rocytes by the method described by Tyler et al. (28) with slight modifications (29). Spectrin dimer was prepared by extraction of human red cell ghosts at 37 “C in low ionic strength buffers followed by gel filtration on Sepharose CL-4B (30). Rabbit skeletal muscle actin was prepared from acetone powder extracts by the method of Spudich and Watt (31). Protein 4.1 and spectrin were stored at 0 “C in 130 mM KC1, 20 mM NaC1, 10 mM Tris, 0.1 mM EGTA,’ 1 mM 2- mercaptoethanol, 30 @M phenylmethylsulfonyl fluoride, pH 7.4 (Buffer I). Actin was stored at 0 “C in 2 mM Tris, 0.2 mM ATP, 10 mM 2-mercaptoethanol, 0.2 mM CaCI,, 0.1 mM phenylmethylsulfonyl fluoride, 0.02% NaNa, pH 8.0 (Buffer A). All proteins were analyzed for purity by SDS-polyacrylamide gel electrophoresis as described by Laemmli (32). Protein concentrations were determined by the method of Lowry (33).

Restricted Proteolytic Digestion of Protein 4.1-Protein 4.1 (at 1 mg/ml) was digested for 30 min in Buffer I, without phenylmethylsulfonyl fluoride, a t 0 “C with a-chymotrypsin at enzyme-to-substrate ratios ranging from 1:200 to 1:400. The reaction was terminated by the addition of 1 mM diisopropylfluorophosphate, and the digests were then either used directly for binding studies or lyophilized in the presence of urea for HPLC separations.

HPLC Purification of Proteolytic Fragments of Protein 4.1-a- Chymotryptic peptides of protein 4.1 were chromatographed on gel filtration columns (two 30-cm Bio-Si1 TSK-400 columns, two 30-cm Bio-Si1 TSK-250 columns, and one 30-cm Bio-Si1 TSK-125 column, all in tandem, Bio-Rad) equilibrated in 8 M urea, 0.2 M Tris, 10 mM 2-mercaptoethanol, pH 7.0. Injection volumes ranged typically from 400 p1 to 2 ml/sample. Effluent was monitored at 280 nm and flow rates of 0.5 ml/min were used. Fractions were pooled and dialyzed against Buffer I.

An active 8-kDa a-chymotryptic fragment of protein 4.1 was further purified by reverse-phase chromatography on a 4.6 X 250-mm C-4, RP-304 column (Bio-Rad) equilibrated with 0.1% trifluoroacetic acid. Elution of the peptide was achieved using a linear gradient of 0

The abbreviations used are: EGTA, [ethylenebis(oxyethyl- enenitri1o)Jtetraacetic acid; HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate.

3310

Spectrin-Actin Binding Site in Erythrocyte Protein 4.1 3311

t o GOr;. acetonitrile in the same buffer. Peptides were detected by absorbance at 215 nm and by tryptophan

fluorescence (excitation 280 nm, emission filter 370 nm). Sedimentation Assa.y.s-Solution conditions for all sedimentation

experiments approximated physiological ionic strength (130 mM KCI, 20 mM NaCI, 10 mM Tris, 0.1 mM EGTA, 0.2 mM ATP, 10 mM 2- mercaptoethanol, 2 mM MgCI?, pH 7.4), where the binding of spectrin to F-actin is minimal in the absence of protein 4.1 (5). Typically, protein 4.1 (0-11 pg) or HPLC purified fragments of protein 4.1, spectrin dimer (33 pg), and G-actin ( 8 0 pa) were mixed to a final volume of 0.1 ml and incubated for 90 min on ice prior to sedimentation. Samples were applied to a 0.2-ml cushion of 10% sucrose in the same buffer and centrifuged a t 0 "C for 30 min a t 150,000 X g in a Heckman SW50.1 rotor. Equivalent portions of supernatants and pellets were analyzed by electrophoresis on SDS-polyacrylamide gradient slab gels (32).

For quantitative hinding studies, spectrin dimer was radioiodinated with the reagent of Bolton and Hunter (34). The activity of protein 4.1, or its active fragment, was assessed based on the amount of '?'I- spectrin cosedimented in a complex with F-actin. Correction for nonspecific binding was made by subtracting the counts sedimented in the absence of 4.1 and actin.

Amino Acid Analysis-Purified chymotryptic peptides of protein 4.1 were hydrolyzed at 110 "C for 20 h in U ~ C U O in 200 p1 of 6 N HCI containing 0.2% phenol. Amino acid analysis was performed on a Dionex D-500 amino acid analyzer using a ninhidrin detection system.

Two-dimensional Cellulose Peptide Mapping-Two-dimensional peptide maps of protein bands from SDS-polyacrylamide gels were prepared essentially by the method of Elder et al. (35) with modifications (36). The gel slices were iodinated using sodium ["'I]iodide (New England Nuclear) and chloramine-T (Sigma). The iodopeptides were digested by tu-chymotrypsin for 24 h a t 37 "C and mapped on cellulose sheets by standard procedures.

RESULTS

Identification of Actiue Fragments of Protein 4.1 Which Promote the Binding of Spectrin to F-actin-Previous work in this laboratory has shown that mild proteolysis by a - chymotrypsin at 0 "C cleaves protein 4.1 primarily in three central locations, producing fragments of approximately 30, 16, 10, and 22/24 kDa (29). In this study we attempted to identify the functional site within protein 4.1 which promotes the association of spectrin and F-actin by examining the activity of a-chymotryptic digests of protein 4.1. A typical digest used for this purpose is represented in the electropho- retic gel profile in the inset of Fig. 2. Although no intact protein 4.1 was seen in these digests, these preparations retained significant amounts of activity in promoting spectrin-F-actin associations. These results are illustrated in Fig. 1. As expected, when spectrin was incubated with F-actin in the absence of protein 4.1, the spectrin remained in the supernatant fraction (lane A ) . In the presence of protein 4.1 both spectrin and protein 4.1 are seen in the pellet with F- actin ( lane B) . An a-chymotryptic digest of protein 4.1 also promoted the binding of spectrin to F-actin (lane C), reflected in the large amount of spectrin seen in this pellet fraction. Note, however, that no a-chymotryptic peptides of protein 4.1 can be detected in the pellet fraction by Coomassie Blue staining. The active components in these digests were identified by separating the peptides by HPLC gel filtration in the presence of 8 M urea (Fig. 2). Fractions, pooled as indicated in Fig. 2, were dialyzed and analyzed for complex-promoting activity by the same sedimentation assay. The results in Fig. 3 show that activity was found in several peaks and could be recovered after gel filtration in 8 M urea. The greatest amount of activity was observed in fractions I and V, which contained primarily 30/32/34- and 8-kDa peptides, respectively. Further analysis of these peaks revealed several peptide components in each fraction.

The molecular weight of the 8-kDa peptide has been determined by HPLC gel filtration in the presence of 8 M urea

Spectrin -

4.1 -

S P S P S P

Actin" - 0 -30

- 22/24 - -16

A B C FIG. 1. Sedimentation analysis of spectrin binding to F-

actin in the presence of intact or digested protein 4.1. Spectrin dimer (33 pg) , muscle actin (30 pg) , and intact protein 4.1 or 4.1 digested with a-chymotrypsin were mixed and incubated for 90 min at 0 "C followed by sedimentation as described under "Experimental Procedures." Equivalent samples of supernatants ( S ) and pellets ( P ) were electrophoresed on a 7-15?; acrylamide gradient SDS gel. Activ- ity was assessed based on the distribution of spectrin between the supernatant and the pellet. A, spectrin and actin alone. R, spectrin and actin in the presence of 6 pg of protein 4.1. C, spectrin and actin in the presence of 16 pg of a protein 4.1 a-chymotryptic digest. Numbers on the right indicate molecular masses of 4.1 chymotryptic fragments in kilodaltons.

I\ I \

(I m

~

0 + I * W 1 4 C I I I 4 WIW b V 4 V

I

30 40 SO

ELUTION VOLUME (ml)

FIG. 2. HPLC gel filtration of a-chymotryptic fragments of protein 4.1. Protein 4.1 (2 mg) was digested with tu-chymotrypsin (1:260 enzyme-to-substrate ratio) a t 0°C for 30 min. The a-chymotryptic fragments were chromatographed on 5 columns in series (Rio- Rad TSK 400, TSK 400, TSK 250, TSK 250, TSK 125; 7.5 X 300 mm each column) a t room temperature and eluted with 8 M urea, 0.2 M Tris-HCI, 10 mM 2-mercaptoethanol, pH 7.0. Fractions were col- lected and pooled as indicated by Roman numerals. Inset, SDS- polyacrylamide gel electrophoresis analysis of the total protein 4.1 digest used for HPLC gel filtration. Numbers indicate molecular masses of peptides in kilodaltons.

3312

- 4.1 S P - -

- Spec t r in -Ac t in B ind ing Site in Erythrocyte Prote in 4.1

+ 4.1 I II III nr Y S P S P S P S P S P S P ”“”

“ ““-“ -

FIG. X. Identification of a-chymotryptic fragments of protein 4.1 which promote binding of spectrin to F-actin. Frac- tions pooled from HPLC gel filtration (Fig. 2, I-V) were combined with spectrin dimer ( 3 3 p g ) and muscle actin (30 p g ) and analyzed by the sedimentation assay under “Experimental Procedures.” Super- natant ( S ) and pellet ( P ) fractions were examined on a 10-1896 SDS- acrylamide gradient gel. Fractions 1 and V showed the greatest activity, reflected in the amount of spectrin in the pellet.

1 51 s

W‘ V z a m a 0 m a u)

J

J 22/24

I I I I

2 0 30 4 0 50

ELUTION VOLUME (ml)

FIG. 4. Isolation of an active a-chymotryptic fragment of protein 4.1 in a ternary complex with spectrin and F-actin. The complex was sedimented from a mixture of 1 mg of protein 4.1 digest, 6 mg of spectrin dimer, and 4 mg of actin as described under “Experimental Procedures.” The supernatant ( S ) and the pellet ( P ) were then separately analyzed in the HPLC gel filtration system described in Fig. 2. The only peptide found within the pellet fraction was the 8-kDa peak. Numbers indicate molecular masses of chymotryptic fragments in kilodaltons.

A S P S P S P ” “ u-

1

48

2 3

I I 0 1 2 3 4 5 8 11

4.1 (pg/lOO pl assay)

FIG. 5. Complex-promoting activity of the 8-kDa chymotryptic peptide. The 8-kDa fragment, isolated in the pellet fraction in Fig. 4, was dialyzed out of the urea, recomhined with spectrin (33 pg) and actin (30 pg) and retested by sedimentation. A, SDS-polyacrylamide gel electrophoresis analysis of spectrin-F-actin complexes sedimented in the absence of protein 4.1 (l) , in the presence of 3 pg of protein 4.1 (2), or in the presence of 1 pg of the 8-kDa peptide ( 3 ) . The 8-kDa peptide greatly enhanced the amount of spectrin that cosedimented with F-actin and can be seen as a faint band in the pellet fraction. R, quantitation of ”‘I-spectrin that cosedimented with F-actin in the presence of protein 4.1 ( O “ 0 ) or the 8-kDa peptide (A-A). The amounts of the 8-kDa f’ragment added are expressed as the molar equivalent of protein 4.1.

under the conditions described under “Experimental Proce- dures,” and verified by the complete sequence analysis of this peptide.’

Isolation of an Active Fragment within a Ternary Complex with Spectrin and F-actin-The experiment shown in Fig. 4 confirmed the identification of an active 8-kDa peptide and enabled the isolation of this fragment from the mixture of peptides. In this experiment, protein 4.1 was digested with a- chymotrypsin more extensively and incubated with spectrin and actin under the conditions described under “Experimental Procedures.” After centrifugation, the supernatant and pellet fractions were analyzed separately by HPLC gel filtration. As

I. Correas, D. W. Speicher, and V. T . Marchesi, manuscript in preparation.


TABLE I Amino acid composition of a-chymotryptic fragments of Protein 4.1

Amino acid compositions were determined as described under “Experimental Procedures” using 20-h hydrolysis only. The number of residues were determined by normalizing the data to fit molecular weights of approximately 8,000, 16,000, and 23,000 daltons for the three peptides. The numbers in parentheses are rounded to the nearest integer.

Amino acid Residues

8 kDa 16 kDa 22/24 kDa Cys“ Asx 7.34 (7) 9.47 (9) 25.00 (25) Thr 0.97 (1) 10.30 (10) 34.06 (34) Ser 7.54 (8) 15.42 (15) 13.65 (14) Glx 11.00 (11) 25.56 (26) 33.88 (34) Pro 3.66 (4) 12.07 (12) 10.02 (10) G b 1.28 (1) 10.37 (10) 13.70 (14) Ala 1.18 (1) 25.00 (25) 13.89 (14) Val 6.72 (7 ) 13.76 (14) Met 2.21 (2) 1.08 (1) Ile 3.59 (4) 2.56 (3) 15.88 (16) Leu 6.04 (6) 4.35 (4) 7.47 (7)

P he 1.93 (2) 1.56 (2) 0.00 (0) His 4.23 (4) 1.76 (2) 5.09 (5)

(l) 0.20 (0)

TYr 0.87 (1) 0.75 (1) 1.19 (1)

LYS 9.23 (9) 10.41 (10) 12.76 (13) Arg 4.84 (5) 11.22 (11) 4.79 (5) Trpb + + -

Cys was not directly quantitated by amino acid analysis; however, its absence was indicated by no reaction with Cys reagents in other experiments.

The presence or absence of Trypt,ophan was deduced by intrinsic Trp fluorescence (excitation 280 nm, emission filter 370 nm) during HPLC separations.

can be seen in Fig. 4, all of the chymotryptic fragments of protein 4.1 were found in the supernatant except one 8-kDa peptide peak, which cosedimented with spectrin and actin. The activity of this peptide was confirmed when fractions from the 8-kDa peak were retested in the sedimentation assay. Fig. 5A shows that the purified 8-kDa peptide retained activity and cosedimented with spectrin and actin. This active peptide was also further analyzed by reverse phase HPLC as described under “Experimental Procedures.” These runs revealed primarily three peptide components, one major and two minor peaks, all very similar in their amino acid composition (data not shown).

A series of binding experiments were conducted to quanti- tate the activity of the purified 8-kDa peptide and compare this with the intact protein 4.1 molecule. The amount of 8- kDa peptide was calculated from amino acid analysis and its activity was compared to the molar equivalent of intact protein 4.1. The determination of binding activity was based on the amount of 125E-spectrin found sedimenting in a ternary complex. Fig. 5B shows that the 8-kDa active peptide pos- sessed activity comparable to intact protein 4.1 on a molar basis. Binding of either the 8-kDa peptide or intact protein 4.1 to the sedimentable complex showed saturation at high concentrations.

Localization of the Active Site within Protein 4.1-Amino acid composition analysis of the HPLC purified chymotryptic peptides of protein 4.1 indicated that the active 8-kDa peptide was not derived by breakdown of the 16- nor the 22/24-kDa acidic peptides. The composition data given in Table I shows that the 16-kDa peptide lacked methionine residues, while the 22/24-kDa peptides lacked phenylalanine and trypto- phane, all three of which were present in the 8-kDa fragment. This suggests that the active peptide was derived either from the basic 30-kDa or the phosphorylated 10-kDa region.

Two-dimensional cellulose peptide mapping was used to establish the location of the 8-kDa active peptide within the whole 4.1 protein. Fig. 6 shows that the active 8-kDa fragment was derived from the 32/34-kDa fragments, since its peptide map produced a pattern which represented a subset of the 32/34-kDa peptides. The 22/24-kDa peptides were also derived from the 32/34-kDa fragments, as has been previously established @9), but none of the peptides in its map coincided with those from the 8-kDa fragment. The maps of the 22/24 kDa peptides and the active 8-kDa fragment represent non- overlapping peptides both derived from the terminal 32/34- kDa fragments. The functional site of protein 4.1 involved in the promotion of F-actin-spectrin cross-linking thus appears to be derived from the protease-sensitive 10-kDa segment previously shown to be phosphorylated by a CAMP-dependent protein kinase (15, 29). These structural relationships are summarized in Fig. 7.

DISCUSSION

Protein 4.1 is one of two proteins that link the spectrin- actin network to the cytoplasmic face of the erythrocyte membrane. Previous work has shown that this interaction requires the filamentous form of actin and both a and p subunits of spectrin (3, 6, 37). This study defines the functional site of protein 4.1 involved in this association. A proteolytic fragment of protein 4.1 of 8,000 daltons was able to promote the binding of spectrin to F-actin when separated from the other a-chymotryptic peptides of protein 4.1. As further evidence that this region of protein 4.1 was involved in spectrin-actin associations, antibodies raised against two different fragments of‘the 8-kDa active peptide were capable of inhibiting this association.2

The location of the active 8-kDa peptide within the 10-kDa domain (see Fig. 7) was supported by both amino acid analysis and peptide mapping. The 8-kDa fragment contained phenylalanine and tryptophan, which precluded a location within the 22/24-kDa domain, and methionine, which precluded a location within the 16-kDa domain (Table I). Analysis of the peptide maps of the active 8-kDa fragment and the 22124- kDa fragments has shown that they were complimentary fragments both derived from the terminal 32j34-kDa fragments.

The functional site of protein 4.1 presented here differs from our previous tentative identification of a spectrin binding site within the basic protease-resistant 30-kDa domain (29) (see Fig.7). This earlier assignment was based on spectrin affinity chromatography and rate zonal sedimentation studies performed in the absence of actin and in low ionic strength buffers (29). More recent experiments have shown that the binding of this 30-kDa fragment to spectrin was nonsaturable, noncompetitive with intact protein 4.1 binding, and was enhanced in lower ionic strength but did not promote the association of spectrin with F-actin. (data not shown). In contrast, the promotion of the spectrin-actin complex by both intact protein 4.1 and the 8-kDa peptide occurred under isotonic conditions. These differences suggest that the weak binding observed between protein 4.1 and spectrin in the absence of actin may be attributable to the unusual polarity in protein 4.1 structure and is probably due to artifactual electrostatic interactions between spectrin and a portion of protein 4.1 enriched in basic residues.

The IO-kDa domain of protein 4.1 also contains the site of phosphorylation by a CAMP-dependent protein kinase (15, 29). The proximity of the spectrin-actin binding site and the phosphorylation site suggests a potential mechanism for reg- ulation of the assembly of these skeletal components and may

3314 Spectrin-Actin Binding Site in Erythrocyte Protein 4.1

34K 24K r 8 K

FIC. 6. Autoradiograms of two-dimensional peptide maps of HPLC purified a-chymotryptic fragments of protein 4.1. Peptides were removed from polyacrylamide gels, iodinated, hydrolyzed with chymotrypsin, and mapped hy standard procedures (35, 36). Electrophoresis was in the horizontal direction while ascending chromatography was in the vertical direction. The arrows show a subset of peptides shared with the map of the active 8-kDa fragment but ahsent from the map of the 22/24-kDa peptides.

30 16 32/34

I I b I fl-l

SHSH SH SH I

P

10 1 22/24

4

P I I

$;. Sp-Actin

FIG. 7. Schematic model of protein 4.1 illustrating the functional site responsible for promoting spectrin-F-actin associations. Kestricted proteolysis by tu-chymotrypsin cleaves protein 4.1 primarily in three central locations, producing four domains of approximately 30, 16, 10, and 22/24-kDa, respectively (29). The basic 3O-kDa domain contains a cluster of cysteines (SH) which are cleaved by NTCB (29). The functional site of erythrocyte protein 4.1 involved in spectrin-actin associations is located within the 10-kDa domain which was also previously shown to contain a site for CAMP-dependent phosphorylation (PI (15, 29). Number.? designate the molecular masses of wchymotr-yptic fragments in kilodaltons. Sp, spectrin.

account for the reported effects of CAMP-dependent kinase stimulation on red blood cell deformability (38-40).

The identification of an 8-kDa fragment of protein 4.1 which is fully functional in the formation of the spectrin- actin complex raises several questions concerning the topo- graphical nature of this ternary complex. Rotary shadowing electron microscopy has demonstrated that protein 4.1-ferri- tin complexes bind to the tail region of spectrin dimers (as), which is also the same region that associates with actin filaments (2). Whether protein 4.1 binds directly to both spectrin subunits and whether direct associations occur between protein 4.1 and actin remains unclear; the active 8-kDa peptide may serve as a useful probe of these linkages. The effect of spectrin and protein 4.1 on the size and rate of growth of actin filaments and their role in stabilizing actin in the form of short protofilaments are presently subjects of intensive study (7-9). While the active 8-kDa peptide binds tightly to spectrin and actin filaments with an apparent affinity similar to intact protein 4.1 (see Fig. 5B), it remains to be seen if this fragment demonstrates the same actin filament “nucleating,” “capping,” or “severing” activities described by others.

Immunoreactive isoforms of protein 4.1, exhibiting a broad range in apparent molecular weights (69-175 kDa), have been identified in a variety of nonerythroid tissues (16-19). The expression of variable protein 4.1 isoforms in avian lenticular and erythroid cells appears to be developmentally regulated (41), suggesting that different isoforms may perform different tissue-specific functions. While these different isoforms have not been characterized functionally it will be of interest to

know which forms possess the spectrin-actin complex promoting activity and to what degree this functional site is conserved.

Acknoudedgments-we thank Drs. Spyros Georgatos, William C. Horne, Richard A. Anderson, and Constantine Axiotis for their critical comments. We gratefully acknowledge the expert technical assistance of Raymond DeAngelis and Anthony Lanzetti. We thank Tina DeGennaro for assistance in typing this manuscript.

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