molecular domain structure of porcine vinculin and metavinculin

Protoplasma (1988) 145:133-140 PROrOPt=l �9 by Springer-Verlag I988

Molecular Domain Structure of Porcine Vinculin and Metavinculin

M. GIMONA 1'*, J. V. SMALL 1, M. MOEREMANS 2, J. VAN DAMME 3, M. PUYPE 3, and J. VANDEKERCKHOVE 3

i Institute of Molecular Biology of the Austrian Academy of Sciences, Salzburg 2 Laboratory of Biochemical Cytology, Division of Cellular Biology and Chemotherapy, Janssen Pharmaceutica Research Laboratory, Beerse 3 Laboratory of Genetics, State University Gent, Gent, Belgium

Summary

Metavinculin is a higher molecular weight variant of vinculin expressed only in cardiac and smooth muscle. Using microsequencing methods on the intact molecules and their proteolytie subfragments we have been able to map the common and different parts of these closely related proteins. Both vinculin and metavinculin, from mam- mals and birds exhibit a relatively protease resistant 90 kD core fragment. N-terminal sequencing analysis of the avian and mammalian core fragments as well as of major core subfragments obtained by extended proteolysis placed the core domain at the N-terminus of the intact molecules and revealed identity between metavinculin and vinculin as well as between species. Limited chymotryptic digestion of porcine vinculin and metavinculin yielded a common 16 kD fragment which could be placed at the C-terminus of the cDNA sequence derived from chick fibroblast vinculin (G. J. PRICE, P. JoNEs, M. D. DAVISON, R. BENDORI, S. GR1FFITHS, B. PATEL, B. GEIGER and D. R. CR~TCHLEY 1988, in press). From additional sequence data the metavinculin specific fragment could be placed at the metavinculin C-terminus. Using a polyclonal antibody specific for porcine metavinculin a peptide unique to metavinculin could be identified. Direct sequencing of this, as well as of related, overlapping fragments, purified by reversed phase HPLC revealed a 68 amino acid insert in procine metavinculin, between the core fragment and the C-terminal piece, common to vinculin and metavinculin. The domain organizations ofvinculin and metavinculin and their possible functional implications are discussed.

Keywords: Difference peptide sequence; Domain structure; Smooth muscle; Specific antibody; Vinculin-metavinculin. Abbreviations." SDS sodium dodecyl sulfate; EDTA ethylendinitri- lotetra acetic acid; HPLC high pressure liquid chromatography.

1. Introduction

The in terac t ions o f cytoskele ta l componen t s with the

cell m e m b r a n e are diverse and complex (GEIGER 1983,

BURRIDGE 1986, NIGGLI et al. 1986, BURN 1988). They

* Correspondence and Reprints: Institute of Molecular Biology of the Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salz- burg, Austria.

m a y involve one or ano the r o f the m a j o r cytoskele ta l

f i laments and be ei ther t r ans i to ry or more p e r m a n e n t

in nature . Par t i cu la r ly no t ewor thy is the occurrence o f

cy to ske l e ton -membrane associa t ions benea th sites o f

con tac t o f cells wi th themselves, with the ext racel lu lar

ma t r ix or wi th synthet ic subs t ra ta . A subclass of these

con tac t sites involves act in f i laments and such sites have

been collectively referred to as junc t ions o f the "ad -

herens t ype" (GEIGER 1983). Since the t rans i t ion o f a

cell f rom a n o r m a l to a t r ans fo rmed pheno type involves

p r ima ry changes in adherens type contacts , leading to

gross changes in cell shape, the molecu la r p a t h w a y f rom

the act in cytoskele ton , t h rough the m e m b r a n e and to

the subs t ra te has been p r o b e d with some en thus iasm

(BuRRIDGE 1986). Nevertheless , we are only at an ear ly

stage in def ining the cascade o f molecu la r l inks and

the mechan i sms tha t regula te their con t ro l led assembly

and d isassembly (BURNS 1988).

A character is t ic c o m p o n e n t o f the contac t s o f the ad-

herens type is the p ro te in vincul in (GEIGER 1983). Since

this p ro te in b inds on the one hand to the act in b ind ing

p ro te in a lpha-ac t in in (BELKIN 1987) and on the o ther

hand to ta l in (MANGEAT and BURRIDGE 1984), vincul in

has been suggested as a l inker between these two mo-

lecular pa r tners in the act in m e m b r a n e cascade (BUR-

RIDGE 1986, GHGER 1983). However , the possible di-

rect in te rac t ion o f vincul in wi th the m e m b r a n e (see

BURN 1988) as well as the existence o f acknowledged

con taminan t s in vinculin p repa ra t i ons (WILKINS et al.

1986, SCHROER and WEGNER 1985, EVANS 1984) in-

dicates tha t o ther molecu la r in terac t ions m a y also exist.

A fur ther interes t ing aspect o f vincul in in terac t ions is

suggested by the existence o f a p ro te in va r ian t o f vin-

culin, namely metavincul in (D'ANGELO SILICIANO and

CRAIG 1982 and 1987, BURRIDGE and CONNELL 1983)

134 M. GIMONA et al. : Molecular Domain Structure of Porcine Vinculin and Metavinculin

that coexists with vinculin in a very tissue specific man- ner (SA~A etal. 1985, GIMONA etal. 1987). Metavin- culin exhibits a higher molecular weight than vinculin and is found only in smooth and cardiac muscle. In this report we review briefly recent work on metavinculin and vinculin (GIMONA et al. 1988, manuscript submitted) that pinpoints the common properties and distinguishing features of these molecules.

2. Materials and Methods

2.1. Protein Purification

Vinculin and metavinculin were purified from porcine stomach or avian gizzard according to a method described earlier (GIMONA et al. 1987) with slight modifications.

2.2. Enzymatic Digestion

Vinculin and metavinculin were digested with either Chymotrypsin, Papain or Staphylococcus aureus V-8 protease (Sigma) under non- denaturing conditions in a buffer containing 20mM Imidazole, 20mM KCI, pH 7.3 at room temperature for 1 to 5 minutes. Re- actions were stopped by adding excess of inhibitors and samples prepared for gel electrophoresis as described below.

(1970) using mini slab gels with a linear acrylamide gradient from 8 to 22%. Samples were supplemented with a buffer (final concentration: 2.5% SDS, 1% 2-mercaptoethanol, 7% glycerol, 0.001% bromphenolblue and 62.5% Tris at pH 6.8) and boiled for 1 minute. Gels were stained with Coomassie brilliant blue G250 (Sigma) and destained in 10% acetic acid.

2.4. Immunoblotting

Silver enhanced immunogold staining was performed according to the procedure described by MOEREraANS etal. (1984) using a secondary antibody with a 20 nm gold tag (Janssen Pharmaceutica, Belgium). The vinculin monoclonal antibody (Vin 11-5; Sigma) was kindly provided by Prof. B. GEmEl~ (Weizmann Institute, Israel). The blotting of minislabs onto nitrocellulose was performed according to Towbin etal. (1979) at a constant current of 40mA for 10 hours.

2.5. Affinity Purification of Porcine Metavinculin Specific Antibody

Polyctonal rabbit antisera against porcine metavinculin were passed over a metavinculin affinity column and the monospecific antibodies eluted with pH changes. The purified antibody fraction was then adsorbed to a vinculin affinity column and the flow through collected. For storage, the antibody fractions were dialyzed against 0.2% su- crose and aliquots freeze-dried in a speed-vac concentrator.

2.3. SDS Polyacrylamide Gelelectrophoresis

SDS PAGE was performed following the procedures given by MATSUDAIRA and BUaOEss (1978) in the buffer system of LXMMLI

2.6. Electron Microscopy

Protein samples were diluted after dialysis in 100 mM ammonium formate, 30% glycerol, 0.5 mM EDTA and 2mM Tris-base, pH 7.1

Fig. 1. Immunoblot of whole tissue samples of different origin. A Human artery; B porcine stomach; C turkey gizzard: D porcine skeletal muscle; E porcine aorta; F porcine esophagus; G porcine uterus; H porcine bladder; I porcine heart muscle; J porcine liver; K porcine spleen; L porcine kidney. Note cross reactivity of monoclonal anti-vinculin antibody with avian, porcine and human proteins

M. GIMONA et al. : Molecular Domain Structure of Porcine Vinculin and Metavinculin 135

to a final concentration of 50 ~tg/ml and sprayed onto freshly cleaved mica according to the procedure given by ELLlorr and OFFER (1978). Molecules were shadowed with platinum at an angle of 6 ~ and sub- sequently coated with carbon in an Edwards E 306 evaporation unit. Electron microscopy was performed on a Zeiss EM 10A at a mag- nification of 25,000.

2.7. Protein Sequencing

Peptides were sequenced on an Applied Biosystems Inc. 740 A gas phase sequenator equipped with an on line 120A phenylthiohy- dantoin amino acid analyzer. Proteins for sequencing were either taken directly after HPLC reversed phase chromatography or from glass fiber sheets coated with poly(4-vinyl-N-methylpyridine) ofelec- troblotted digests. Details on the blotting procedure are given by VANDIZKERCKHOVE etaI. (1987) and BAUW etal. (1987).

3. Results

3.1. Tissue Distribution o f Vincutin and Metavinculin

The general ubiquity o f vinculin and the restricted expression o f metavinculin are revealed in immunoblo ts

o f tissue extracts with non-discriminat ing vinculin-metavinculin antibodies (Fig. 1: see also SAGA et aL 1985,

GLUKHOVA etal. 1986, GIMONA etal. 1987). Meta- vinculin is specifically expressed in smooth muscle

(Fig. 1 A-C, F-H) and cardiac muscle (Fig. 1/) and is

absent f rom adult skeletal muscle (Fig. 1 D) and non-

muscle tissues (Fig. 1 J and L). The origin o f the sur-

prisingly large amounts o f metavinculin in spleen (Fig. 1 K) remains to be clarified but appears not to result f rom contamina t ion with vascular smooth mus-

cle. As noted previously (GIMONA et aL 1987) the rel- ative amounts o f vinculin and metavinculin vary con-

siderably among different smooth muscles, even in the

same species (compare Fig. 1 B, E, F-H), metavinculin being predominan t in s tomach (B), esophagus (F) and

bladder (H).

In smooth muscle cells vinculin and metavinculin antibodies label submembranous a t tachment plaques

(GEIGER etal. 1981: Fig. 2A, inset) that form contin-

"uous rib-like arrays parallel to the cell axis (Fig. 2 B, SMALL 1985). Whether or not vinculin and metavin-

culin are exactly co-distributed in these surface ribs has not yet been established.

3.2. Molecular Morphology

After ro tary shadowing, avian vinculin and metavinculin exhibit a characteristic and identical head and

Fig. 2. A (upper left inset): Cross section of guinea pig taenia coli smooth muscle stained with a nondiscriminating polyclonal vinculin antibody showing the characteristic dotted vineulin distribution along the cell periphery. B Isolated t. coli cells stained with the same antibody revealing continuous surface ribs along the axis of the ceils

136 M. GIMONA et al.: Molecular Domain Structure of Porcine Vinculin and Metavinculin

tail morphology in the electron microscope (Fig. 3 A 1." MOLONY and BURR~DGE 1985, GIMONA etal. 1987). Multimeric aggregates of these molecules are also commonly observed and appear to arise from side- to-side and end-to-end associations between the tail regions (Fig. 3 A 2-4). Under the same conditions the corresponding molecules from porcine stomach appear only globular in shape, lack a tail region and do not readily aggregate (Fig. 3 B and C, GIMONA et al. 1987). In view of the otherwise close cross-species identity of these molecules (see below) this apparent difference in molecular morphology is surprising.

3.3. The Common 90 kD, N-terminal Core Domain

Limited proteolysis of avian and mammalian vinculins and metavinculins yields a relatively protease-resistant "core" fragment (Fig. 4A and E; FERAMISCO etal. 1982, G~MONA etal. 1987). N-terminal sequence analysis of the vinculin and metavinculin core fragments and the intact molecules revealed complete identity for at least the first 20 amino acids (GIMONA etal. 1988, manuscript submitted) and identified the core as the N-terminal part of both proteins. Recent sequence analysis of a chick fibroblast vinculin cDNA (PRICE etal. 1987) showed corresponding identity of these smooth muscle sequences with those of avian non-muscle vinculin. Extended proteolysis of the 90 kD core fragment from porcine vinculin and metavinculin yielded major subfragments of 67 kD and 39 kD whose N-terminal sequences could be aligned precisely with the fibroblast vinculin sequence. The cleavage sites yielding these fragments approximately delineated the three repeated

Fig. 3. Rotary shadowing of vinculin and metavinculin molecules. A selection of avian vinculin molecules showing the head and tail structure of the molecule (1) and the possible modes of aggregation via association of the tail regions (2-4); B porcine vinculin and C porcine metavinculin lack the tail exhibited in the avian molecules. Arrowheads indicate individual molecules. The contrast in C is pho- tographically reversed. Bar, 10nm

Fig. 4. Enzymatic fragmentation of porcine vinculin (A and B) and metavinculin (C-E) showing the different characteristic fragments. A V-8 protease; B Chymotrypsin; C Chymotrypsin; D Papain; E V-8 protease

motifs already noted from the avian vinculin sequence, corroborating conclusions (PRICE etal. 1987) about the existence of a repeated, triple domain structure in the core regions. Further peptide analysis of HPLC purified core fragments chemically cleaved in 80% formic acid, revealed more or less complete cross-species and cross-variant identity within the avian and mammalian vinculin and metavinculin cores. These data identify the core as a highly conserved region of these molecules.

3.4. The C-terminal Region of Vinculin and Meta-

vinculin: Identification of the Difference Peptide in Por- cine Metavinculin

Limited proteolysis of porcine vinculin and metavinculin yielded a series of low molecular weight fragments (Fig. 4) whose N-terminal sequences did not match any of those in the core domain (PRICE et al. 1987). These peptides could thus be identified with the C-terminal part of the molecules. Slightly extended chymotryptic digestion of porcine vinculin and metavinculin yielded similarly-sized 16 kD fragments (data not shown) with identical N-terminal sequences: these fragments could both be aligned with the derived C-terminal part of fibroblast vinculin, starting at residue 913, some 50 amino acides beyond (in vinculin) the

M. G1MONA etal.: Molecular Domain Structure of Porcine Vinculin and Meta-dnculin 137

starting at the N-terminus of the 30 kD fragment and terminating at the cleavage site of the 16 kD peptide. Further peptide analysis of the 26/28 kD vinculin doublet (Fig. 4A) and a comparable 14/16kD doublet in metavinculin (GIMoNA et al. 1988, manuscript submitted) showed that the N-terminus of the 30 kD fragment corresponded to a c o m m o n chymotryptic cleavage site in vinculin and metavinculin and constituted the true start of the metavinculin difference peptide. A schematic diagram of the vinculin and metavinculin molecules based on this data is shown in Fig. 6 and the amino acid sequence of the difference peptide in Fig. 7. Further sequence data is presented elsewhere (GIMONA et al., manuscript submitted). Worth noting is the presence of a pair of characteristic K W S S K sequences, flanking both sides of the metavinculin difference peptide and that contain the native chymotryptic cleavage

Fig. 5. Characterization ofmetavincutin specific antibody, m Marker lane; a coomassie blue gel of chymotryptic metavinculin digest with the major fragments of 105 and 30kD; b corresponding immunoblot showing reaction only with the C-terminal 30 kD fragment and the remaining intact molecule but not with the core-containing 105 kD fragment. Molecular weight markers from top to bottom: 200, 150, 130, 92, 68, 57, 51, 42, 31, and 20kD respectively

9okD 16kD

end of the core region. The size of the vinculin 16 kD fragment and the position of its N-terminus suggested that it extended to the true C-terminus of the intact molecule. Further sequence data (see below) indicated that the similar metavinculin peptide likewise constituted the far C-terminal part of metavinculin. The key to the identification of the peptide unique to metavinculin was provided by the production of an antibody specific only for metavinculin. This was obtained by the adsorption of a polyclonat metavinculin antibody on affinity columns of metavinculin and vinculin. When used in immunoblots of limited digests of metavinculin (as in Fig. 4 C) this antibody reacted ex- clusively with intact metavinculin and the chymotryptic 30 kD fragment (Fig. 5). I t failed to react with the 20 kD and 24 kD peptides produced by cleavage with V-8 protease and papain, respectively, as well as with the 16kD chymotryptic fragment ( immunoblot not shown). Extended N-terminal sequence analysis o f the 30, 24, and 20 kD fragments revealed a sequence overlap of these peptides, in that order, as well as an overlap of the 20 kD sequence into the 16 kD C-terminal peptide, common to vinculin and metavinculin. These sequences delineated a 68 amino acid difference piece

N

90kD

C

KW SSK KW

3okD

24kD

Pap V-8 Ch Ch

SSK

Fig. 6. Molecular domain structure of porcine vinculin (upper model) and metavinculin. N-terminal 90 kD region (including the three in- ternal repeats R 1, R2, and R3), the proline rich sequences (P) and C-terminal 16kD fragments are common to both proteins. Meta- Vinculin differs from vinculin by a 68 amino acid insert (i) that contains additional cleavage sites for papain (Pap) and V-8 protease (V-8). Chymotrypsin recognition sites delineate the beginning and the end of the insert inside the flanking KWSSK sequences. The overlapping 30, 24, and 20 kD fragments extend from the insert into the C- terminal 16kD fragment (arrows)


KWSSK /~X~/C-terminus

T

Loops

Epitope K H S S K P G N P A R K V G I G V W E R D R R D n V G F

P V P S D M E D D Y P E L L L M P S S Q P V N Q P I L R A

A q S L H R E R T K W S S K

Fig. 7. Secondary structure of the metavinculin specific insert as predicted from the primary sequence. The flanking KWSSK sequence (straight arrows) are most likely to form beta turns with the N-terminal sequence followed by a helix (Helix 1) consisting of 8 amino acids. 3 loops connect this helix to Helix 2 which is then again followed by a turn formed by the KWSSK flanking region. The encircled P and the bent arrow indicate the position of the predicted tyrosine phosphorylation site. The amino acid sequence of the insert is shown below in one letter code. The epitope of the metavinculin- specific antibody iies within the first 25 residues. Connected open arrows indicate the two helix regions, the asterisk marks the position of the tyrosine within the putative recognition site for tyrosine kinase

sites (for the 30 kD and 16 kD fragments) between the Trp(W)-Ser(S) residues. A corresponding, single KWSSK sequence occurs in vinculin and contains the cleavage site for the common 16kD fragment. The position of insertion of the difference peptide is sited beyond a proline rich linking region between the core and the C-terminus (see als PRICE etal . t987) that is strictly conserved in the different vinculin and metavinculin variants studied. Within the metavinculin difference peptide itself several potential phosphorylation sites could be identified, some containing serine and a single site with a consensus substrate sequence of a tyrosine kinase (HUNTER and COOPER 1985).

4. Discussion

The strict conservation of the core domain between the vinculin and metavinculin variants, as well as between species and in muscle and non-muscle cells points to

this region as an essential, functional part of these molecules. BURRIDGE and colleagues (MOLONu and BURRIDGE 1985, MANGEAT and BURRIDGE 1984) have already shown that the core domain possesses the talin binding site and corresponds, apparently, to the globular head piece noted in the electron microscope (MoLoNV 1985). On this basis the head piece can be identified with the N-terminal region of avian vinculin and metavinculin. Experiments with isolated, ventral membranes of fibroblasts have indicated that vinculin is not readily saturable in these contact regions (GEIGER et al. 1985) and this has been taken as indicative of vinculin-vinculin association in vivo. According to the appearance of the multimeric aggregates observed after rotary shadowing such associations would most likely occur through tail-tail binding, that is through the C-terminal parts of the molecules. Evidence has also been obtained for the binding of alpha-actinin to vinculin and metavinculin (BELKIN etal . 1987) and for the specific association of vinculin with acidic phospholipids (NIGGLI etal . 1986). More recently, phospholipid binding has also been observed for porcine vinculin and metavinculin (NIGGLX, manuscript in preparation). The pre- ferred association with phosphatidyl serine likely occurs in the hydrophobic, proline rich region common to both vinculin and metavinculin (see Fig. 6). The sites of binding of alpha-actinin to vinculin and of other proposed vinculin associated proteins (see, e.g., BUR- RIDGE 1986, NIGGLI and BURGER 1987) have yet to be established. D'ANGELO-SILICIANO and CRAIG (1987) have reported an eight fold greater phosphorylation of avian metavinculin as compared to vinculin. Although we have no information on the molecular composition of the difference piece in avian metavinculin we have identified several characteristic serine doublets as well as a tyrosine containing region in the difference peptide of porcine metavinculin, consistent with the presence of potential multiple phosphorylation sites in this region. Identification of the difference peptide in avian metavinculin has been hindered by the lack of cross reactivity of the porcine metavinculin antibody and by a different pattern of proteolysis of avian as compared to porcine metavinculin. We presume that these reflect minor differences in primary structure that lead to different cleavage characteristics. Further sequence data will be needed to clarify this point. F rom a consideration of the primary structure of the metavinculin difference peptide we propose a secondary structure as depicted in Fig. 7. This structure con-

M. GIMONA et al. : Molecular Domain Structure of Porcine Vinculin and Metavinculin 139

tains two short alpha-helices interspaced by a triple loop region. The position of the putative tyrosine phosphorylation site is also shown. The positive labelling of the 30 kD chymotryptic metavinculin fragment and negative reaction of the 24 kD papain and 20 kD V-8 fragments by the metavinculin specific antibody places the epitope within the first 25 N-terminal residues of the metavinculin insert (Fig. 7). The noted identity of metavinculin with vinculin outside the region of the difference peptide is consistent with these two proteins arising by alternative splicing at the mRNA level. To understand the co-existence of vinculin and metavinculin in rive more data are needed on the molecular interactions that distinguish between these molecules. Recent ideas about protein-lipid interactions (BURN 1988) raise the possibility that associations of membrane lipids and proteins may play a role in the regulation of cytoskeleton-membrane interactions. It is not inconceivable that the association of lipids with the proline rich linkage region adjacent to the position of the metavinculin insert, could induce conformational changes that may modify, for example, the access of tyrosine kinases to their target sites. Likewise the possibility exists of associations of specific molecules with only metavinculin directly via the difference peptide or as a result of conformational differences induced by its presence. Future studies are aimed at further clarifying the molecular partners involved in the cytoskeleton- membrane cascade.

Acknowledgements We thank Prof. B. GEIGER for providing a monoclonal vinculin antibody (vin 11-5; Sigma) and G. J. PRIC~ and colleagues for making the complete sequence of chick fibroblast vinculin available to us before publication. This work has been supported in part by a grant from the Austrian Science Research Council and by a Post Graduate Fellowship (to M. G.) from the Boehringer Ingelheim Fund. J. V. is Research Associate of and was supported by a grant of the Belgian National Fund for Scientific Research.

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molecular domain structure of porcine vinculin and metavinculin

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