91

Btochlmtca et Blophystca Acta, 1031 (1990) 91-110 Elsevier BBAREV 85361

Cell surface receptors for extracellular matrix components Steven K. Akiyama

L2, K a z u h i r o

Nagata

3 and Kenneth

M. Yamada

2

I Departments ofOncology andBiochemtstry, Howard Umoerstty Cancer Center, Howard Unwerslty College of Medicine, Washington DC and : Membrane Bwchemlstry Secnon, Laboratory of Molecular Btology, National Cancer Insntute, Nanonal Insntutes of Health, Bethesda, MD (U S .4 ), and 3 Chest Disease Research Insntute, Kyoto Unwerstty, Sakyo-ku, Kyoto (Japan) (Recewed 5 October 1988) (Revised manuscript recewed 22 June 1989)

Contents I

91

Introduction

92 92 95 96 99 101

F~bronectm receptors A General properues B The avmn flbronectm receptor C Mammalian 140 kDa flbronectm receptor D Platelet glycoprotem IIb-IIIa complex E Glycoprotem Ic-IIa complex III Vltronectm receptors

101

IV Collagen receptors

102

V

104

Larmmn receptors

VI Conclusions

106

Acknowledgement

106

References

106

!. Introduction Cellular interactions with basement membranes and extracellular matrices are crucial for a number of biological processes that reqmre cell adhesion or migration including wound heahng, embryogenesis, the maintenance of tissue integrity, and the metastasis of cancer cells Adhesive and migratory events not only require that cells attach to extracellular materials, but also involve interactions of xntracellular cytoskeletal proteins with membranes (cf Ref. 29). One of the major challenges of cell biology and biochemistry is to elucidate the macromolecular structures involved in cell adhesion

Abbrevlauons VLA, very late antagen, TGF-fl, transforrmng growth factor-fl, MNJ, 1-deoxymannojmmycm Correspondence S K Akayama, Department of Oncology and Btochermstry, Howard Umverslty Center, Howard Umverslty College of Medicine. Wasbangton, DC 20060, U S A

and nugratlon, and then to understand their interactions and regulation. Intracellular proteins that are involved in cytoskeletal function (e.g., actin, a-actinm, vinculln and tahn) as well as m a n y extracellular adhesive proteins such as the collagens, fibronectlns, lamlnln and vltronectin have been fairly well characterized In contrast, the integral membrane molecules that act as receptors for the extracellular adhesive proteins and that provide putative transmembrane links to lntracellular cytoskeletal proteins were until recently either unknown or very poorly characterized. In the past few years, however, there have been several breakthroughs that have yielded a great deal of information on receptors for adhesive proteins In this review, we shall focus on the eukaryotlc cell receptors for fibronectm, vltronectln, lamtnln and the collagens. This revaew is selective rather than exhaustive. Due to space hmltatlons, we have chosen to emphasize topics based primarily on our own interests

0304-4157/90/$03 50 © 1990 Elsevier Science Publishers B V (Biomedical Division)

92 Those readers who seek coverage of other topics are urged to consult some of the other excellent recent revaews on this subject (e g, Refs 25, 26, 103, 120, 129, 201 and 202)

TABLE 1

Llgand

Cell type

K d (/z M)

Receptors per cell

Refs

II. Fibronectin receptors

Flbronectm Fibronectm CBD a F]bronectin

fibroblasts

8 10 7

1 4 - 5 105

1,3

hepatocytes platelets

1 7 10 8 3 10 7

2 8 105 1 2 105

126 187

Collagen type I

fibroblasts

1 2 10 H

5 105

83

tarmmn

carcinoma melanoma fibrosarcoma

(2-4) 10 -9

0 1-1 ] 105

153, 195, 231

H-A General propernes Flbronectin is a multlfunctlonal glycoprotem found m extracellular matnces and body fluids that can medmte the adhesion and magratlon of various cell types (reviewed in Refs. 6 and 159). Fibronectln monomers are monomers of approx 250 kllodaltons (kDa) in s]ze which form disulfide-bonded dxmers and sometimes larger multimers There are several related forms of fibronectm The two major forms are found m blood (plasma fibronectin) and secreted locally onto cell surfaces and into extracellular matnces (cellular flbronectm) Most studles use plasma fibronectin, which is a soluble dimer consisting of very similar but not identical polypeptide chains The biological acUvmes of fibronectIn are contamed in a senes of protelnase-resistant domains, which have been individually punfied and characterized A schematic model of the human plasma fibronectln molecule showing the relauve sizes and locations of the domains of fibronectin is shown in Fig 1 (reviewed in Refs 6, 121, 159 and 244) Even before the ldentlflcauon of specific receptor molecules, the interactions of cells with fibronectin were charactenzed using cell attachment, spreading and direct binding assays Although cells spread very efficiently on substrates coated with microgram amounts of fibronectm, the fibronectln-receptor xnteractmn is of only moderate affinity ( K d = 8" 10 -7 M) [3], much lower than the affinities observed for other extracellular matrix protein receptors (Table I) There are 140 000 to 500000 surface fibronectin receptors per cell [1,3] Cell spreading and direct-binding assays have been used to compare the actlvaty of intact fibronectm with

H,N--~

}"1

}J1

Iv1

Cell bmdmg parameter~ o/ e-~tracellular rnatrt ~ protem~

a 85 k D a RGDS-contaming cell-binding domain of fibronectm

purified proteolytlc fragments of flbronectin In both systems, the smallest fibronectln fragment that retains full binding act]xnty is a 75 kDa fragment that encompasses the central one-third of the fibronectm polypeptide [1,99] This fragment appears to contain all the information required for the adhesion of fibroblastic cells [1,99,167]. A smaller 12 kDa fragment has cell attachment activity [180] and can inhibit the binding of fibronectln to cells, but it has 10-100-fold lower binding affinity [3] A key cell binding site of fibronectln has been localized to a short segment of four amino acid residues with the sequence arglnlne-glyclne-aspartic acld-serlne (RGDS) [4,181-183,246] Synthetic peptldes containing the R G D S sequence can block the adhesion of cells to fibronectin, inhibit binding of soluble fibronectin to cells, and, under certmn conditions, promote the adhesion of cells However, a second, distant, functionally synergistic site within the cell-binding domain is also needed for recogmtion by cells to mediate efficient adhesion and cytoskeletal organlzauon [167]. Fibronectin receptors that interact with the cell-binding dommn of fibronectin have been isolated from axaan and mammahan tissues and cells. These 140 kDa fibronectln receptor systems each appear to contain two non-cova-

VA

: ~

35k 30k

40k

20k

HzN=-I I-"1 I.,f'l ~ Heparln Collagen Flbnn Ftbnn Gangllomdes

75k VA Cells

~ C O O H 30ks/s/ ////

Co0k SS ." I~1 I ] [[COON Hepann Flbnn

Fig 1 The domain structure of flbronectin A current model of the d o m m n structure of plasma fibronectm is shown It consists of two smular but nonidentical glycoprotem chams connected by two disulfide bonds The rectangles represent the individual protemase-resistant, globular binding domains, wbach are connected by less structured regions of the polypeptlde chain represented by wavy hnes The approxamate sizes of the domains are indicated along v,ath a short hst of their respectwe hgands The approramate posluons of three d~stlnct cell recogmtlon sites are also shown the R G D S site (dotted hne), a second site for fibroblastic cell binding acuvlty (hatched), and separate cell recogmtton sites for neural crest derivatives (black)

93

09' I f

PLASMA MEMBRANE

\ Fig 2 Model of the structure of the fibronectm receptor m t e g n n s Detergent-solublhzed receptor visualized by electron microscopy appears to have a globular head with two tails on the same side [32,130,164] wluch probably are anchored in the plasma membrane Both the a and fl subunlts contain membrane-spanning regions, so both are shown as contnbutlng one tad each and part of the head The a subumt of these receptors ~s composed of two polypepudes that are disulfide bonded just outside the plasma membrane, and contains three or four calcium binding repeats The fl s u b u m t contains four cysteme-nch repeats and extensive m t r a c h a m disulfide bonding

lently bound subunits. The larger component, termed the a subunlt, usually ranges in slze from 140 to 165 kDa The smaller fl subunit is usually 120 to 130 kDa m size The avian receptor is tsolated as complexes of three glycoprotelns wtth molecular masses of 120-160 kDa When visualized by a variety of electron-rmcroscoplc methods, purified, detergent-solubihzed receptors appear to be composed of a globular head with two relatively long legs or tails [32,130,164] If detergent lS not present, the receptor complexes form clusters or 'rosettes' by aggregation of the taft regtons, suggesting that the tatls are hydrophobic and normally anchored m the plasma membrane. A highly schematic model of the hypothetical structure of the visualized receptor is shown in Fig. 2. The avian and mammalian 140 kDa flbronectln receptors belong to a large family of related glycoproteln complexes termed 'integrlns'. The lntegrin farmly also includes the platelet glycoproteln IIb-IIIa complex and the vltronectin receptor The lntegrins share similar heterodlmenc structures conslstmg of an a and fl subunit noncovalently associated tn a 1 : 1 ratio [105,106]. So far three major (and two minor) classes of integnn fl polypeptides and 11 different integrln a polypeptldes have been identified. The various a polypeptides are related and are probably derived from a common evolu-

tlonary precursor, as are the fl polypeptldes, however, the as and fls are not closely related to each other Hynes [120] has proposed the deflmtlon of three different classes of mtegrlns based upon the different fl subunlts. As shown in Table II, the/31 lntegrlns include the avian flbronectln receptor, the human flbronectln receptor, at least one collagen receptor, and the T-cell 'very late antigen' (VLA) heterodlmers [106,120,225227,238] Members of this family contain one of six (or more) unique but related a subunIts with a molecular mass of 140-210 kDa associated with the common /31 subumt in a noncovalent dlmer Clearly, the a subumts must play a role in confernng specificity on the individual complexes by as yet unknown mechanisms All of the fll integrins that have been characterized have functions associated with cell adhesion For example, a3fl 1 is contained in the avian flbronectin receptor [106,225], and asfl I is the mammahan flbroblast fibronectin receptor [11,225,226] The ct subunlts of the fll subfamily that have been sequenced are 21-42% identical at their armno termini [225] These regions in ct subumts from aafl I (chicken receptor) and t~sfl1 (human receptor) are 42% identical [225] The fll subunits from different species are evolutionarlly conserved to a high degree For example, avian and human fll polypeptldes have 85% amino acid identity, with identical carboxy-terrmnal sequences [11,228], and anubodies that recognize the cytoplasmic domain of the chicken fli subumt identify proteins from nematodes and fungus [154]. Furthermore, the human fll has an 82-86% identity m anuno acid sequence with two different/31 mRNAs from Xenopus laevls [57]. The class 2 mtegrlns are leukocyte-specific and contam at least three different a-polypeptldes associated with the common fiE subunIt This class includes the LFA-1, Mac-l, and p150,95 heterodlmers [10,144]. The primary structure of the human f12 polypeptide is 47% identical with the chicken fll subumt [144] There are at least two possible a polypeptldes associated with the f13 subumt These heterodimers form the vltronectm receptor and the platelet glycoprotein IIbIIIa complex which brads flbronectln, fibrlnogen, von Willebrand factor, and wtronectm A fourth type of fl subunit ~s found m some vltronectin receptors, and a fifth appears to mediate certain types of neutrophll interaction with RGD-contalnlng adhesion proteins [44,88,104]. Components of the avtan and mammalian flbroblast fibronectin receptor, as well as the platelet glycoprotein IIb-IIIa complex, and the vltronectin receptor have been cloned and sequenced at the cDNA level. The properties of specific a and fl polypeptides derived from c D N A sequencing are summarized in Tables III and IV The fl polypeptldes analyzed to date have a small carboxy-terrmnal intracellular domain, a transmembrane region, and a large extracellular domain that

94 TABLE I1

The mtegrm famdy of adheston receptors Abbrewatlons Coil, collagen, Fn, hbronectm, Ln, lamlnm, Fb, fibrmogen vWF, von Wdlebrand factor, Vn, vltronectm. ~lsp, thrombospondm Name

Alternauve names

1 The fll m t e g n n s (VLAs) al fll VLA-1 a2 fll VLA-2, GPIa_ Ha ECMR! ct3fl 1 VLA-3, ECMRII Ot4fl 1 VLA-4 ctsfl 1 VLA-5, fibronectm receptor, GPIc_ lla a6fll VLA-6

L~gands

Putatwe adhesion function ~

Arb~trard'~ ~elected Refs

Coil, other ° Collagen types I-IV, VI Fn, Ln, Coll Fn, other9 F~bronectm

Cell-matrix Cell-matrix

137 219,224,238

Cell-matrix Cell-cell and cell matrix Cell-matrix

32 225 110,239,224 8,11 70

Larmnm

Cell-mamx

216

2 The f12 m t e g n n s (leukocyte adhesion receptors) OtLfl 2 LFA-1 CtMfl2 Mac-l, CR-3 a x f12 p150,90

I-CAM-l, I-CAM-2 C3bl 9

Leukocyte cell-cell Leukocyte cell-cell Leukocyte cell-cell

10 l0 10

3 The f13 m t e g n n s (cytoadhesms) ailbfl 3 GPilb_liia

Fb, Fn, vWF, VN

81,176

avfl 3

Vn, Fb, vWF, Tsp

Platelet cell-cell and cell-matrix Cell-matrix

202,215

4 (Alternative vltronectln receptor) etvfl4 Vltronectm receptor

Fn, Vn

Cell-matrix

44A04

5 (Phagocytosls-enhancmg receptor) a.fl~ Phagocytoslsenhancing receptor

Fn, Fb, Vn, vWF, coll type IV

Sttmulatlon of neutrophd phagocytosls

88

Vltronectm receptor

Provisional hstmg - m m a n y cases, further detmled analyses wdl be reqmred In a d d m o n , classtficauon as presently uncertain for awan fl~ receptor bands 1 and 2, awan smooth muscle mtegrm a s u b u m t [130], as well as the Drosophda myospherold gene product [146,152]

extracellular domain has 56 cystemes and a cluster of 4 cysteme-rlch repeats which contains extenswe lntracham dlsulhde bonding The a subunlts also contain a small carboxy-termlnal lntracellular domain, a transmembrane reglort, and a large extracellular domain that contains the anuno

contains the amano terrmnus The lntracellular domain often contains a sequence homologous to the tyroslne phosphorylauon s~te of the epidermal growth factor receptor, and both the m a m m a h a n and avaan receptors from transformed cells have been reported to be phosphorylated m the presence of vanadate [108,236] The

T A B L E Ill

Compartson of mtegrm a subumts a

N u m b e r of arrano acids b A m i n o acids m transmembrane segment A m i n o acids m cytoplasmic domain N u m b e r of cystemes Potential N-linked glycosylatlon sites Putative Ca-binding repeats

Fibronectm receptor

Collagen receptor

G P Ilb

Vltronectm receptor

Drosophda PS2 A n u g e n

~t 5

Ot 2

Ot[i b

Ot v

Ot

1 008 29 28 20 14 5

1152 22 22 20 10 3

1008 20 28 17 5 4

1017 29 32 18 13 6

1 363 26 30 15 12 4

a Data from Refs 11, 12, 16, 64, 70, 71, 190, 221,222 and 224 b Excluding signal sequences

95 T A B L E IV

Comparison of mtegrm fl subumts a

N u m b e r of armno acids b Amino acids in transmembrane segment Armno acids in cytoplasnuc domain N u m b e r of cystemes Potential N-hnked glycosylatlon sites N u m b e r of cysteme-rlch repeats

Avian fli

Mammalian fll

GP Ilia

Xenopus

Drosopht~

fll

fl

777 23 47 54 12 3

778 23 47 58 12 4

762 29 41 56 6 4

777 23 47 56 11 4

823 23 47 56 6 -

Data from Refs 11, 70, 71,152 and 228 b Excluding signal sequences a

terminus along with a series of divalent c a u o n - b m d m g domains. The a subumt often consists of two disulfide hnked polypeptldes, the smaller chmn contains the transmembrane and lntracellular domains The a subunits have evolutionanly conserved amino termxm, but are not related to each other to the same degree as the various fl subumts [120]. H - B The avtan ftbronectm receptor The avaan lntegnns including the flbronectln receptor have been isolated from homogenates of cultured chicken fibroblasts, myoblasts and whole chicken embryos by immunoaffimty chromatography with monoclonal antibodies [34,89,98,163] and from adult chicken smooth muscle using biochemical methods [130] As

M r xlO -a

155 135 120

m

i

Band 1 Band 2 Band 3

Fig 3 Avaan hbronectm receptor Flbronectln receptor was lmmunoprectp~tated from metabohcally labeled cluck embryo hbroblasts and analyzed by gel electrophoresis and fluorography as described [5] The receptor appears as three components termed band 1, band 2 and band 3 m order of decreasing size

shown in Fig. 3, this receptor system appears as three glycoproteln components with molecular masses of 155-165 (band 1), 135 (band 2) and 110-120 kDa (band 3) [39,98,133] Band 1 may have a small 25 k D a disulflde-hnked component. The three bands are distinct polypeptldes w~th no immunological cross-reactivity [98,130,133]. Band 3 is immunologlcally crossreactive with fl subunlts of the other members of the fll (VLA) family [225]. Bands 1 and 2 probably contain a variety of distinct a polypepudes The hydrodynamic properties of punfied receptor suggest the presence of complexes the size of dlmers, and anti-band 1 monoclonal antibodies isolate only bands 1 and 3 (Refs 25, 27, 98 and 112; Yamada, S S, unpublished data) These results imply the existence of two distract hetero&menc complexes consisting of bands 1 and 3, and bands 2 and 3 The functions of the individual components of the avaan receptor complex are still unknown. The binding of flbronectin requires band 3 in a complex w~th at least one of the other subunits - neither bands 1 and 2 together nor band 3 alone can bind to fibronectm [28]. The interaction between flbronectm and detergentsolublhzed avian receptor Is of low to moderate affinity [9,112] The receptor is retarded and not bound when passed through a column of immobilized flbronectm [9]. Although weak, this interaction has been quantitated ( g d = 10 -6 M) using a gel filtration method [112]. Current evidence suggests the avian flbronectin receptor is a multlfunctional receptor or a mixture of related receptors. Monoclonal antibodies that bind to the receptor inhibit cellular interactions with flbronectin, lanumn, vltronectln and collagens [25,38,50,55,112]. More importantly, interactions of avian receptor with larmnln and vltronectm have been demonstrated directly [25,112]. L a n u n m binds with relatively low affinity - K d = 2 10 -6 M [112]. Vitronectm inhibits the binding of both larmmn and fibronectin to the receptor [21], suggesting that lanunm, vitronectln and flbronectm are interacting with either the same site or closely related sites [25,112]. The mechanisms involved m the

96 binding of the various adhesive protein hgands to the avian 'fibronectm receptor' will be important to resolve At present, it is probably prudent to refer to this complex as the avian fli mtegrin complex rather than simply the fibronectin receptor. The avian integrin receptor complex also binds to the cytoskeletal protein tahn ( K d = 7- 10 -7 M), but not to a - a c t m m or vmcuhn [111] The binding site for tahn on the receptor complex is on the intracellular portion, distinct from that for fibronectin Likewise, the receptor binding site on tahn is distinct from that for vmcuhn [25] These data suggest that the avian lntegrln receptor can directly link the cytoskeleton in the interior of the cell with extracellular matrix components on the outside of the cell via tahn The avian mtegnn (fibronectin) receptor appears to be a substrate for tyroslne phosphorylatmg enzymes under some conditions Bands 2 and 3 of receptor from Rous sarcoma virus-transformed chick embryo fibroblasts m culture can be labeled on tyrosme with [32p]_ orthophosphate in the presence of vanadate [108], while receptor m untransformed cells is not The phosphorylated receptor binds neither fibronectin nor talln [25] Nevertheless, the physiological relevance of the phosphorylation of the receptor IS unclear at present It is only observed after treatment of the cells with vanadate [108], an agent that can affect cells directly [155] Furthermore, the receptor from embryomc neural crest cells and motde somatic cells is not phosphorylated, yet, both of these cell types resemble transformed cells in their lack of an organized cytoskeleton and cell-surface fibronectln as well as their high motility [61]. In contrast, receptors from mature somltlC cells that phenotypically resemble normal fibroblasts are phosphorylated The transmembrane nature of band 3 has been demonstrated directly, using monoclonal antibodies separately directed against the extracellular and intracellular domains [160] and also using polyclonal antibodies rinsed agmnst synthetic peptldes corresponding to the cytoplasmic domain [154] The lntracellular domain partially co-distributes with extracellular flbronectin [160]. The locahzatlon pattern of lntegrln (flbronectin) receptors m the plasma membrane of cultured cells is consistent with receptor involvement in cell adhesion and motdity Integrlns on migrating cells are diffusely distributed over large regaons of their surfaces [40,53,63], and are highly mobile [62] On stationary cells, the receptor is organized Into ~mmoblle aggregates that appear to be associated with the ends of extracellular flbronectm fibrils, intracellular actln mtcrofilament bundles m a linear assocmt~on with a-actlnin and fibronectln, and m or around focal contacts, suggesting that the receptor can act as a link between the lntracellular cytoskeleton and the extracellular matrix

[38-40,53,163,202] Early studies agreed that the a~mn receptor was enriched about the periphery of focal contacts but excluded from the center [39,53], although this result is not found in m a m m a h a n cells Other work has locahzed avian lntegrln epltopes to the centers of focal contacts [130], suggesting differences in location or exposure of different epitopes, these confusing issues still need resolution Mahgnant transformation of chick cells in culture results in the loss of receptor-containing transmembrane structures [40] along with the loss of cell surface fibronectln and cytoskeletal organization The transmembrane fibronectln-contalning complexes can be restored by the addition of high concentrations of exogenous cellular fibronectm [40] A similar disruption of the pattern of the cell surface fibronectin receptor can be observed upon the addition of high (though nontoxic) concentrations of GRGDS-containing fibronectln-hke peptldes The results suggest that fibronectm receptor distribution can be regulated by its hgand, i e, fibronectm (Ref. 40, see also Ref 199) The expression of/31 lntegrm (fibronectln) receptors Is increased in Rous sarcoma virus-reduced tumors in VlVO, compared with uninvolved adjacent tissue [203] The receptor appears to be diffusely distributed m tumor cell plasma membranes, consistent with observations made with transformed cells m culture [40,77] The role of the increased expression of various mtegrm receptors in tumors will be important to elucidate, e g, m permitting tumor cell rmgraUon In fact, antibodies against the avian receptor can block cell invasion across a basement membrane [210] Studies with synthetic peptldes also suggest that a fibronectm-type receptor may play a role in metastasis [118] It is thus possible that increased receptor expression might be a more general requirement or even a marker for some invaslve cancer cells

H-C Mammahan 140 kDa fibronectm receptor The m a m m a h a n flbronectln receptor consists of a 145-155 k D a a and a 125 k D a /3 subunit (see Fig 4) Both subunlts have been cloned and sequenced (see Tables III and IV) [11,12]. A schematic model of the structure of the human flbronectln receptor is given in Fig 5 The ct component is a disulfide-bonded complex of a large peptxde and a small 25 kDa peptlde [11,106,127] Both peptldes orlgmate as part of a single large polypeptide that ~s post-translatlonally cleaved Neither the mechanism of this proteolytlc processing of the flbronectm receptor nor its function is known Mammalian fibronectm receptor can be affinity purified directly on columns of immobilized flbronectm after detergent extraction using procedures developed by Pytela et al [191,193]. Receptors have been punfled from human osteosarcoma cells [193], hepatocytes [127],

97

M r x l 0 -s

145-125 --

~4

--

alpha

--

beta

Fig 4 Mammahan flbronectm receptor Flbronectm receptor was lmmunopreclpltated from surface-lodmated cultured mouse 3T3 cells and analyzed by SDS gel electrophoresls and fluorography as described [5]

phagocytes [20], lymphocytes [149], keratmocytes [235], placenta [191], rat fibroblasts [193], hamster fibroblasts [21,22], murine fibroblasts [5], erythroleukemla cells [78,174] and thymocytes [31]. The fl subumt has also been purified from mouse tumor tissue, using classical blochelmcal techmques [230]. The hepatocyte receptor appears to be functionally

distinct from the fibroblast receptor, suggesting the existence of cell-type specificity [127]. The hepatocyte receptor binds RGD-contammg fragments of fibronectm with a 50-fold higher affinity than the flbroblast receptor [1,127]. Furthermore, antibodies that recogmze the hepatocyte receptor inhibit spreading on fibronectln and not on larmnln [127], whereas both monoclonal ant~-fl and polyclonal antibodies that recognize the fibroblast receptor mhlbit fibroblast spreadmg on both fibronectm and larmnm [8,236]. Hepatocyte and fibroblast receptors probably contain distract a subumts and a common fl subunlt [127] It is not yet known to which member of the fll subfarmly the hepatocyte fibronectm receptor corresponds At least two types of flbronectln receptor can co-exist on certain cells The asfl ~ class can be isolated by Its capacity to brad to fibronectm affinity columns asfl ~ binding to flbronectxn appears to be specific, both by hposome-bmdmg cntena, where it does not bind to lanunln or vltronectln [193], and by immunological inhibition criteria, where it binds specifically to flbronectin compared with collagen or lanumn [8,21,22,33]. In contrast, the ot3fl 1 class (ECMR I) binds weakly to fibronectm, it appears to display multiple binding actlvrues dubbed 'pron'uscuous' by some workers, 1.e., it interacts with fibronectm, collagen and lamlmn accordmg to immunological criteria and by analogy to the avian receptor system [33,225-227] In addmon, it lS hkely that other fibronectln receptors may be found. Certain cells, such as some derived from the embryomc neural crest specifically recogmze alternatively sphced sites m the IIICS region of fibronectm not bound by other cells (e.g., see Refs. 116 and 117) The role of calcium with the flbronectln receptor is not yet clear Calcium is not required for the initial stages of cell attachment [90], for the binding of fibronectm-coated beads [91], or for the binding soluble fibronectm [3] to cells However, isolated, detergentsolubihzed fibronectm receptor cannot bind hbronectm after treatment wath EDTA [191], although the subunlts

Ca binding repeats I

Extracellular •

1Rr

HzN-(.

HzN-(.

Cytoplasm=c

1



VV

4MM~

V/,,-~//./YJ'//~//~ ~ •

J



COOH

A A A

=

Cys-nch repeats

H

Plasma membrane Fig 5 Domain structure of the mammahan flbronectm receptor Proposed structure of the mammahan fibronectm receptor based on the sequence data m references [11,70] The a chain consists of two polypeptldes, which are disulfide-bonded near the transmembrane region Both the a and fl chains contain a small mtracellular carboxy-terrmnal domain (horizontally stnped), a transmembrane segment (black), and a large extracelhilar domain (white) The approxtmate relatwe posmons of the putative calcmm b m & n g domains (sUppled), the cysteane-nch repeats (hatched), the potential sites of asparagine-lmked glycosylatlon (v), and a possible site of tyrosme phosphorylauon ( t ) are also given

98 PULSE-CHASE LABELING OF FIBRONECTIN RECEPTOR SUBUNITS

CHASE TIME (hr) 0

A

4

8

12 28

Band 1 Band 2-Band 3--

g

alpha-- i m ~ ~ j ~ beta-- i l ~ l l ~ D m pre b e t a -

C

alpha _ beta -pre b e t a -

Chmken fibronectm receptor

~

Mouse fibronect)n receptor

Human flbronectln receptor

Fig 6 Biosynthesis the flbronectm receptor from cultured clucken, mouse and human cells Cells were incubated for 2 h with [35S]methiomne and then m medmm lacking radmactwe label as described [5] At the mdmated times, cells were lysed and the flbronectm receptor was lmmunopreclpltated and analyzed by gel electrophoresls followed by fluorography The cluck cell receptor contmns three &stmct protein components The human and mouse receptors contain two &stmct protem components The smaller fl chain is mmally synthesized as a smaller precursor (pre-fl) that requires an unusually long time for lntracellular processing At mtermedmte Umes (4-20 h after chase), three bands are observed wtuch correspond to mature a, mature B and pre-fl At long times after chase (greater than 22 hours), only two polypepttdes are observed, winch correspond to the mature a and fl chains

appear to remain associated (Yamada, S S , unpublished data). Clearly, the flbronectm receptor is at least stablhzed or protected by calcium Receptor function is maintained after treating cells w~th protemases in the presence of calcium, whereas receptor function ~s lost after treating cells in the absence of c a l c m m [3,169] Thus, ~t will be ~mportant to analyze systematically the effects of calcmm on the structure and functmn of the fibronectm receptor in experiments analogous to those described below with the platelet G P I I b - I I I a receptor. T h e / 3 subumt of the m a m m a h a n fibronectln receptor appears to undergo unusually slow lntracellular processing [5,124,197] As shown m Fig 6, all three components of the chicken receptor and the a - c o m p o nent of the m a m m a h a n receptor undergo lntracellular maturation that is complete m 1 - 4 h [5,124,197] However, the /3 c o m p o n e n t of the m a m m a h a n receptor ~s mmally synthesized as a smaller precursor form that is only slowly processed into a larger, mature form result-

lng .n the loss of mannose and the incorporation o! glucosanune [5] This process requires 20 h for complet]on (t 1 , = 4 h) [5,197] Importantly, the processing o! the/9 subumt to its mature form results m the acqmslu o n of ftbronectm-blndlng acuvlty of the receptor complex [5] Th]s maturatton of the fl subumt (and also the o~) involves a conversion of ohgosaccharldes from endoglycosldase H-sensmve to endoglycosldase-H resistant forms (Akiyama, S.K and Yamada, K M , unpubhshed data) The biosynthesis of the m a m m a h a n fibronectm receptor by cultured cells can be modified by treating the cells with transforrmng growth factor-/9 (TGF-/9), which Increases the a m o u n t of both the a a n d / 9 components of the flbronectln receptor m cultured mouse [124] and h u m a n cells [197] by increasing the a m o u n t of m R N A , new p r o t e m synthes]s, and altering the rate of receptor processing [124,197] The maximal effect on m R N A levels is observed after 24 h of treatment [197] In mouse cells, the a and p subunits are increased 6and 8-fold, respectively [124] In h u m a n cells, the a m o u n t of receptor complex is increased only 2-fold m the presence of serum, but the increase is 20-40-fold m the absence of serum [197] The increase of flbronectm receptor is not necessarily correlated wtth increased proliferation of cells [124] TGF-/9 has broad effects on the synthesis of other m t e g n n receptors it substantially increases levels of c~ subumts 1, 2 and 3, as well as the vltronectm receptor and LFA-1 [102,123] Although de novo biosynthesis of receptor requires 2 0 - 2 4 h [5], flbroblastlc cells trypslnlzed m the absence of calcium recover their ability to spread on flbronectm substrates w]thln 3 0 - 6 0 man [94,96], suggesting the presence of a substantml mtracellular pool of spare receptors EsUmates of the size of the mtracellular pool of receptor range from 50% to 99% of total cellular flbronectm receptor [22,156], although another study m a different cell type found httle or no mtracellular pool of receptor [23] The nature of the signal required for the secretmn of fibronectm receptor has not yet been determined Preliminary results mdmate that the bulk of the mtracellular pool of receptor consists of immature/9 subumt [5,7], suggesting the hypothesis that cells modulate receptor secretion by regulating the m a t u r a t m n of covalently b o u n d o h g o s a c c h a n d e s However, cultured cells treated with the Golgl mannosldase mhxbitor 1-deo x y m a n n o j m m y c m ( M N J ) synthesaze subunlts of the fibronectm receptor that are not processed into mature forms, but are nevertheless assembled into c~-/3 complexes and secreted into the plasma m e m b r a n e [7], apparently excluding the involvement of N-hnked ohgosaccharldes m the receptor secretion signaling process Identlflcatmn of the flbronectln receptor secretion stgnal wall be important to deterrmne and may have general lmpllcatmns for the regulatmn of the cell surface expressmn of other receptor systems

99 Synthesis of fibronectm receptors in mahgnant mammahan cells has been analyzed with varying conclusions. A variety of rat fibroblastlc cells were found to synthesize much less fibronectxn receptor, as well as two other fll lntegnns [186]. In contrast, mahgnant human fibroblast denvatxves showed no such alteration m a or fl subumts of the flbronectm receptor, but instead revealed a marked increase m rate of biosynthetic processing of the fl precursor [2] These results compare w~th the increase of avian mtegrlns in wvo and no change m v~tro [40,203]. Thus, although it is clear that mahgnancy can alter the metabohsm of mtegrm receptors, the specific changes may be tumor- or speclesspeofic. Freshly explanted keratlnocytes cultured from unwounded skin do not express functional cell surface flbronectln adhesion receptors [95,235], although keratmocytes isolated from heahng wounds do [95] Functional receptor eventually appears on the surfaces of cultured keratmocytes after 10 days in culture, suggestmg that culture condmons may resemble those of a heahng wound The freshly explanted keratmocytes reportedly contain an mtracellular 50 kDa fragment of the fibronectm receptor, suggesting that newly synthesized receptor polypeptldes that are not secreted are degraded lntracellularly This interpretation is conSlstent with the observation that approximately twothirds of newly synthesized 13 polypeptldes are degraded mtracellularly before secretion m human lung fibroblasts [197], and suggests that mtracellular degradauon of 'excess' receptor polypeptldes could help regulate cell surface expression of the receptor The localization of the fibronectm receptor on cultured human cells appears to be dependent on culture conditions and adhesive substrate. Receptor appears to accumulate m focal contacts, m streaks co-ahgnmg w~th stress fibers [77,92,213], and colocallzang w~th substratebound fibronectm [92] on cells that are fully spread on fibronectm substrates m the absence of serum. When cells are m the process of spreading and on cells that spread m the presence of serum, the receptor is concentrated more m close- or matrix-contact sites [77,213] Puzzhngly, m some cell types but not m others, receptor can also co-accumulate w~th vltronectm receptors on vltronectln substrates, suggesting either incomplete speclfioty or receptor-receptor interactions [56,213] There is also a subpopulatlon of receptor that is diffusely distributed on the dorsal surfaces of the cells In a manner similar to the awan fibronectm receptor, mahgnant transformaUon of mammalian cells results in the replacement of the &screte dxstnbuUon pattern of receptor by a more diffuse pattern [2,77] Functions of the hlgh-affimty asfl 1 fibronectm receptor have recently been probed by monoclonal antlbo&es [8,199,238] Although capable of medmtmg lnltml attachment of normal fibroblasts, the receptor is not

essential for subsequent cell spreading or cell rmgraUon on fibronectm [8]; we speculate that these functions may be mediated by different receptors stdl remaimng to be characterized, e.g, ~t3fl1 or vltronectm receptors. Instead, the flbronectln receptor functions to slow the rate of normal flbroblast rmgratlon, and its mhtbmon stimulates cell locomotion. The receptor is centrally involved m the orgamzatlon of fibronectm fibrils on the cell surface AnUbodles against either subunlt mlublt the retention of fibronectin and its assembly into a matrix [8] Other receptors probably also contribute to this process [159] In addition, the formation of speclahzed cell contacts with the substrate and actm nucrofilament bundle orgamzat~on also appear to be at least partially dependent on receptor function [8] Antlbo&es against the receptor also cause its redlstrlbutxon to a diffuse pattern resembhng that on transformed or highly nugratory cells [8,199] All of these findings are consistent with the hypothesis that tills fibronectm receptor plays a central role m the transition of cells from the highly migratory to the stationary phenotype. Thus, rapidly nugratmg or tumor cells tend to display a diffuse, mobde organization of receptor, retain fibronectm poorly, and show minimal numbers of focal contacts and nucrofilament bundles As cells become more stationary, e.g, during differentiation after cell mlgrat~on, fibronectln receptors form streak-hke, immobile aggregates on the cell surface, and the cells estabhsh specmhzed cell-substrate contacts, form extracellular flbronectln matrices, and orgamze mtracellular rmcrofllament bundles (reviewed m Refs 8 and 62) F~bronectm receptor function may also be regulated by other plasma membrane constituents, although the mechamsms are still obscure. Ganghosldes can regulate the capacity of cells to organize a fibronectm matrix, and occupancy of collagen receptors can block fibronectm receptor function for cell spreading (reviewed m Ref 244).

H-D Platelet glycoprotem lib-Ilia complex Platelet adhesion and aggregation are essential for survaval by providing hemostasls after trauma Platelets funcUon by adhering to several large, adhesive plasma glycoprotelns including fibrinogen, flbronectm and Von Wxllebrand factor [81,128,148,176,177] The platelet glycoprotem complex termed 'IIb-IIIa' (GP IIb-IIIa) acts as a receptor for these adhesion proteins Peptxdes containing the fibronectm recognition sequence R G D S and also the fibnnogen gamma chain decapepude LGG A K Q A G D V can lnl~blt the binding of these adhesive proteins to both platelets and purtfled GP IIb-IIIa [75,80,172,173,188,209,243]. The G P IIb-IIIa complex is a 1 . 1 hetero&mer of the glycoprotelns IIb and IIIa [32,179] The GP IIb-IIIa

100 complex must be intact to bind to adhesmn proteins [69,113,140,213]. When viewed as a member of the mtegrln receptor superfarmly, IIb corresponds to the t~ subumt and I I I a to the /3. Because of its unusual abundance compared with other members of the superfamily (1-2% of total platelet protein), the G P I I b - I I I a complex can be purified in functional form in mllhgram quantities from human platelets using relatively simple sucrose density centrlfugatlon or lectln affinity chromatography methods [68,69] The avaxlabdlty of such large amounts of purified receptor has greatly faclhrated blochermcal analysis of the GP I I b - I I I a complex. Calcium is required for platelet adhesion, for hgand binding to purified GP IIb-IIIa, and also for maintainmg the integrity of the native GP I I b - I I I a complex [69,73,140,170-172,177-179,260] Direct calcium-bradmg studies indicate the presence of two high-affinity sites ( K d = 9 nM) and six low-affinity s i t e s ( K d = 400 nM) per G P l i b - I l i a complex [18] Lowering the calcium concentranon to less than 0 01 nM causes a reversible dissoclanon of the lib and IIIa subumts Reconstitutlon of either calcmm or manganese within 10 man results in reassocmnon of the active receptor complex, but magnesium is ineffective [69,73,140,250] However, in the continued absence of divalent cations, the dissociated subumts aggregate irreversibly, eventually preventing reconsntunon of active complex In fact, subunlts d~ssoclated by the removal of calcium appear to be unfolded [32,69], and calcium appears to regulate the surface exposure of an epitope near the carboxy-termlnus of the G P lib large chain [80,151] These studies suggest that calcium is reqmred for both s u b u n l t - s u b u m t interactions (in contrast to the mammalian fibronectin receptor, winch does not dlssocmte after calcium removal), and for maintaining the conformation of individual subumts G P I I I a as present on endothelial cells as part of the vltronectln receptor, wluch can mediate the attachment of these cells to adhesive proteins [35,37,41,165,232], and GP IIb is present in erythroleukemm cells Using m R N A from these nucleated cell sources, G P lib and GP IIIa have been cloned and sequenced at the c D N A level [70,71,190] G P IIb has a polypeptlde molecular weight of 120 kDa, which leaves 18 k D a of the mature subumt to be accounted for by post-translational modifications such as glycosylatlon. There are five potential sites for asparagme-hnked glycosylation A glycosylated precursor is cleaved to yield the final disulfide-bonded G P IIb subunxt compnsed of a large chmn of 110 k D a that contains the original amino terminus and a carboxy-terminal transmembrane chain of 18 k D a [19,64,70,190] The nature and funcnon of the proteolytic processing step is unknown, and it may be important to elucidate m the future The small chain contains a 26 amino acid residue transmembrane sequence, a 20 residue cytoplasmic domain, and one of

the potential N-hnked glycosylatlon bites, its presence an the G P I I b - I I I a complex is not required for binding to R G D pepndes [143] The larger chain is entirely extracellular It contains four of the five of the potennal glycosylatlon sites and four repeating domains consistmg of about 30 amino acids that are homologous to the calcium binding sites of troponin C and calmoduhn, and therefore represent the hkely sites for at least some of the calcium interaction sites on this receptor [70] G P IIb is homologous with c~ subunlts of other receptors in the mtegrm superfamlly It has 38 and 36% sequence identity with the a subumts of the fibronectln and vltronectm receptors, respectively Interestingly, the vltronectln a subumt can replace G P IIb m the GP I I b - I I I a complex to form a functional vltronecttn receptor [11,36.37,70,145,190], and platelets actually express both receptors on the cell surface [142] G P I l i a has a polypepnde chain of 90 kDa and 15% carbohydrate [70 71] Like the GP IIb subunlt, it has a large amino-terminal extracellular domain with several potential sites for asparaglne-hnked glycosylatlon and four cystelne-rlch repeats There ~s a 29 amino acid residue transmembrane segment near the carboxylterminus, leaving only a small mtracellular domain GP I l i a is identical to the vltronectm receptor B subunit [82] and contains striking sequence homologies to /3 subunlts from the fibronectm receptor and leukocyte adhesion molecule (LFA-1) G P I I I a has 47% sequence identity to the avian fibronectm receptor /3 subumt, 44% to the human fibronectln receptor fl subunlt, and 39% to the LFA-1/3 subunlt [11,71,131,228] On resting (inactive) platelets, the G P I I b - I I I a complex is present on the cell surface, but m inactive form [69] U p o n activation with thrombm or ADP, the G P I I b - I I I a complex is rapidly activated to a form that can bind fibrlnogen, fibronectln, von Wlllebrand factor and vltronectln The mechanism of this activation remains to be elucidated Activation-enhanced binding of RGD-contalning peptldes and the 12 kDa fragment of fibronectin has been observed in crosshnklng studies [60,74,208]. The 12 k D a fragment was found to crosshnk to GP IIIa [74] In studies with synthetic peptides, R G D pepndes were preferentially crosshnked to G P I I I a [60] or to both GP IIb and G P I I I a [208]. The R G D binding domain was defined further by determining the sequence in G P I I I a located in closest proximity to the crossllnked R G D pepndes [59] This site consists of residues 109-171 in G P I I I a , a segment that is conserved (76% sequence identity) among the /3 subumts of the integrlns [59] This region may therefore play a crucial role in the adhesive functions of the integnn family Activation-dependent changes in conformation a n d / or rmcroenvironment of this receptor have been detected with monoclonal antibodies [51,72] Activation also causes a change m the platelet-surface d~stribution

101 of GP lib-Ilia from diffuse to clustered [125] This clustenng effect appears to be caused by receptor occupancy Small R G D S contaimng peptides and also the fibnnogen "t chain decapeptlde L G G A K Q A G D V can cause clustering of activated G P IIb-IIIa [125]. This clustenng event and the alterations in epltope exposure may be related to known alterations in association of the receptor with cytoskeletal elements. The binding of synthetic flbronectln and flbrlnogen peptldes can also result in direct conformatlonal changes in purified GP IIb-IIIa [173]. These changes are reflected m an increased susceptlblhty to proteolysls of GP IIb, decreased intnnslc fluorescence and sedimentation coefficient, and increased Stoke's radius, consistent with a hypothesized peptide-lnduced partial unfolding of the complex [173] Whether these changes are related to the cell surface clustenng observed with occupied GP IIb-IIIa will be important to determine in the near future

H-E Glycoprotem Ic-IIa complex Resting platelets prior to activation can also attach and spread on fibronectln in a specific interaction with the RGDS sequence. Adhesion to flbronectm substrates by these non-activated platelets is mediated by a 140 kDa receptor system identified in fibroblasts which corresponds to the platelet glycoprotein Ic-IIa complex [79,105,185,216,245]. Under non-reducing conditions, G P Ic and GP IIa are 135 and 120 kDa, respectively. There are small amounts of at least GP IIa, which crossreacts with the /3 subunit of the fibroblastlc flbronectin receptor, on the surfaces of non-activated platelets. The GP Ic-IIa complex functions in platelet adhesion to flbronectln independently from the GP IIb-IIIa complex. Antibodies against the fibroblastlc fibronectin receptor block the adhesion of non-actwated platelets to flbronectin, while antibodies against GP IIb-IIIa have no effect [185]. In contrast to the G P IIb-IIIa complex, but hke the flbroblast fibronectin receptor, GP Ic-IIa can function even in the absence of calcium [185] The coexistence in platelets of both an activatable and a weaker, continually acUve adhesion system may permit their attachment to exposed subendothehum while avoiding aggregation until the time is appropriate. Although both may be important under certain conditions, R G D peptlde inhibition studies suggest that yet a third mechanism of adhesion to

fibronectin is revealed under the high shear conditions of rapid blood flow [166]. III. Vitroneetin receptors Vltronectln (serum spreading factor) is an adhesive plasma glycoproteln that mediates the attachment and spreading of cultured cells [15,100,109]. Vltronectinmediated spreading occurs independently of flbronectin-mediated spreading. Like fibronectln, vltronectln has binding sites for collagens, glycosamlnoglycans, and cells (see Fig 7) The cell binding site contains an R G D sequence that binds to its receptor [192,194] In fact, the vltronectln receptor binds to the R G D sequence with higher affinity than the flbronectin receptor Detergentsolubihzed vltronectin receptor binds directly to lmmoblhzed RGD-contalnlng synthetic peptldes and can be eluted with excess soluble peptlde as a complex of two glycoprotelns of 125 kDa (a) and 115 kDa (fl) [194]. Surprisingly, the vltronectln receptor isolated in this manner binds specifically to vltronectln and RGDcontaining synthetic peptldes but not flbronectin [191,194] The mechamsm of the specificity of the flbronectln and vitronectln cell adhesion receptors is still a matter of some controversy The two simplest hypotheses are that receptor specificity is conferred by (1) a site on the adhesion protein distant from the R G D site [167] or (2) the conformation of the polypeptlde chain around the R G D site [184] Whether the secondsite hypothesis or the conformatlonal hypothesis (or some combination of the two) is correct will be important to determine in the near future. At present, some conformatlonal restraints seem important for R G D recognition by the vltronectin receptor family (GPIIb-IIIa and vltronectln receptor), since a cycltzed R G D peptide and tngraman, an RGD-containlng peptlde, are more active than hnear R G D peptides, and conformation-specific antl-RGD-reglon peptides have been raised [47,114,134,184]. However, flbronectln recognition appears to require a distinct second region elsewhere in the protein [167] The vltronectln receptor binds calcium and the ganghoside GD2 [43] Calcium is reqmred for association of the ganghoslde with the receptor and for the RGD-bindlng function of the receptor [43]. This effect appears to be calcium-specific, since reconstltuuon of EDTA-treated receptor with magnesium will not restore activity. The binding activity of the vltronectm receptor

5k

H

z

N Cell SornatornedmB

~ Collagen

~

12k C O Hepar)n

O

H

F~g 7 Schematic model of the structure of vltronectm Vltronectln consists of a single polypeptlde chain with speofxc binding sites for collagen (stippled), cells (black), and glycosalmnoglycans (hatched) The armno-terlmnal sequence of vatronectln is identical to the serum protein somatomedln B

102 is also enhanced by adding exogenous ganghoslde G D 2 with calcium, although adding ganghoslde without calcmm has no effect [43] Like the flbronectln receptor, the precursors of the and fl chains of the vItronectin receptor contain high m a n n o s e ohgosaccharides However, lntracellular processing of both chains of the vltronectm receptor is essentially complete after 8 h and so, occurs much more rapidly than for the flbronectln receptor [5,42] The significance of these differences is not yet known Assembly of the a and fl chains occurs very early in the bmsynthetic process [45] Newly synthesized a chain binds to vltronectm peptide affinity columns within 7 mIn after biosynthetic pulse-labeling, well before final maturation of the ohgosacchandes and simultaneously with association to B chains [45] However, fl chains require 60 man to be chased into c~-fl complexes [42] Thus, newly synthesized c~ chmns must associate with 'older' fl chains, probably out of an accumulated lntracellular pool [45] In the absence of a chains, free 13 chains are not functional and are not inserted Into the plasma membrane, but instead accumulate lntracellularly [45], suggesting that assembly of the a-13 complex is required for both biological activity and insertion of the receptor into the plasma membrane It is not yet known if ol chains are functional m the absence of fl chains By using radlolabeled chemacal crossllnkers coupled to RGD-contalnlng synthetic pepUdes, the R G D binding site on the vitronectm receptor has been localized to the regmn between 61 and 203 amino acid residues from the amino terminus of the fl subunit [215] This regmn is highly homologous with a similar region In the flbronectln receptor [11,215] Therefore, although it has not yet been proven, it is highly hkely that this sequence may define a common integrln bmdlng site for R G D containing adhesive proteins The locahzatlon of the vitronectm receptor on cultured cells suggests that it plays a role in cell adhesion and mxgrauon On cells m the process of spreading in serum containing cultures, vltronectln receptors colocalIze with flbronectln receptor and m focal contacts [43,213] On well-spread cells, vltronectm receptor remains localized in focal contacts [43,56,213] A novel vltronectm receptor has been identified recently on carcinoma and other epithelial cells, which consists of the same a-subunit associated w~th a new fl-subumt distinct from any known lntegrln [44,104] This receptor can mediate cell adhesmn to vltronectm as well as fibronectm, but not to flbnnogen or von Wlllebrand factor, a pattern of recognition distract from that of prewously described wtronectln receptors [44]

IV. Collagen receptors Collagens comprise a large family of multlmenc structural glycoproteins, at least 13 distinct types of

collagen have been identified Some ol the collagen types (such as types I and II) exist as large fibers, while others (such as type IV) form non-flbrlllar structures Collagen was initially thought to be a stable but relatively inactive constituent of connective tlssue~ It has become lncreaslngl~y obvious that collagens play important roles In morphogenesls, development, metastasis of cancer cells, and cellular differentiation As with the other extracellular matrix molecules discussed m this review, the effects of collagen on cells are thought to be mediated through specific cell surface receptors In contrast to the other receptors, many different putative collagen receptor molecules have been identified, but none have yet been characterized in detail As was the case for fibronectln, the presence of specific cell surface receptors for collagen was first inferred indirectly by cell attachment [93,211] and direct binding assays using soluble collagen [83] Soluble, radlolabeled type I collagen binds to a single class of binding sites on flbroblasts with high affinity ( K d = 1 2 10-11 ) This interaction is specific as judged by inhibition studies using collagen fragments or collagen-specific antibodies [84,236] Identification of possible collagen receptors has been accomplished by (1) examining detergent-solublllzed molecules that bind to collagen immobilized on Sepharose, (2) identifying proteins that bind to insoluble collagen fibrils, and (3) examining molecules that are recognized by antibodies that inhibit cell attachment to collagen. Table V summarizes the putative collagen receptors that have been reported. These receptors can be divided into two groups according to size The group A receptors are smaller and monomerlc, ranging in size from 31 to 70 k D a The group B receptors are larger and can be multimenc, with components m the range of 90 to 250 k D a Of the group A collagen receptors, the best characterized is anchorin C | I This receptor is a 31 kDa glycoprotein purified from chick chondrocytes by affinIty chromatography on immobilized native type II collagen [158] The corresponding mammalian receptor has been purified from sheep flbroblasts and has a molecular mass of 34 k D a [157] The avian and mammalian anchorins are immunologlcally crossreactive [157] Anchorln CII can bind to native chick collagen types I, I1, III, V and M [158]. Interestingly, the detergentsolublhzed anchorln CII is dissociated from collagen type II by 50-100 m M NaC1, but anchorln reconstituted into hposomes will bind to collagen type II even in the presence of 0 5 M NaCI Anchorin CII has been cloned [67] and found to consist of 329 annno acid residues, its predicted molecular mass is thus 37 344 kDa, winch is larger than the size estimated from electrophoresis [158]. Furthermore, no N-terminal signal sequence was found, and there are three charged residues within the putative transmem-

103 TABLE V

Collagen receptors S~ze (kDa)

Cell type

How ~solated

Remarks

Refs

Group A 31

cluck chondrocytes

Affimty column b

34 65 61

sheep fibroblasts h u m a n platelets h u m a n platelets

158 157 30 135

47-65

mouse endoderm cells

Affimty column a Affimty column a Banding to insoluble collagen a Affimty column c d

A n c h o n n CII Brads to types I - I H , V and M Related to a n c h o n n CII Brads to a 1 chain Transmembrane 47 k D a protein not on cell surface

141

70

Affimty column ac Affimty column a

K d = 2 3 10 -8 M

40

cluck corneal eplthehal cells rabbit spermatozoa

220 136

Group B 20-140

rat hepatocytes

Affimty column a

Antibodies lnlubtt adhesion to collagen

200

Monoclonal antibodies Monoclonal antibodies

a3fl I

238

135/155

h u m a n fibrosarcoma cells h u m a n platelets

90-120 75/80 90

cluck embryo fibroblasts h u m a n platelets h u m a n fibroblasts

Affinity c o l u m n " Affimty column Monoclonal anUbodxes

125/145

Idenucal to G P I a - I I a complex and azfl 1 Idenucal to factor XIII Colocahzes with vlmentm

139 168 205 33

" Type 1 collagen b Type 1I collagen Type IV collagen d Gelatin

brane region. These findings suggest that the molecule may not be an integral membrane receptor; m fact, It shows considerable homology with members of the calpactm/hpocortln family, which are cytoplasmic proterns that can bind calcmm and phosphohplds The protein is not processed, which is consistent with its lack of a signal sequence and N-11nked glycosylatlon, yet it is somehow released into the culture medium of fibroblasts and chondrocytes [175] It Is not yet clear how much is perxcellular versus lntracellular; the overall role of tills protein m cell lnteracUons with collagen obwously requires further clarification. Other collagen-binding proteins have been isolated from platelets by affinity chromatography on collagen a I chains [30] and by binding to insoluble collagen fibrils [135]. The former receptor candidate has a molecular mass of 65 kDa and brads to collagen type I with an affimty constant of 5.107 M -1 [30]. The latter receptor candidate has a molecular mass of 61 kDa [135] It ~s not yet clear whether these two receptors are tdentical or distinct molecules Collagen-binding proteins of approx 47 kDa m size have been ~solated using affimty chromatography [141,162,220]. However, this protein could not be locallzed to the cell surface and IS now known to be a novel heat-shock protein located in the endoplasmlc retlculum [161,204]

Sugrue [220] has also purified a 70 kDa collagen binding glycoproteln from eplthehal cell membranes using either lmmoblhzed denatured type I collagen or native type IV collagen. When reconsmuted into hposomes, it brads to either denatured type I collagen or native type IV collagen with a K d of 2 3 10-8 M. The larger-sized group B collagen receptors have been identified more recently. By isolating monoclonal antibodies that lnlublt specific cell adheswe functions, Wayner and Carter [238] have characterized two different collagen receptors that are member of the class 1 mtegnn family. One receptor, termed ' E C M R I', is a heterodlmer containing polypeptldes of 147 kDa (a) and 125 kDa (fl) that brads to collagen and also fibronectxn and lan~nln. A second heterodlmer (termed E C M R II) has a 145 kDa a subunlt associated with the same B subumt as E C M R I, but bands only to collagen ECMR I and E C M R II have been identified as ct3fl1 and a2fll, respecUvely [227]. The reportedly collagen-specific receptor, a 2/~l,lS also ldenUcal with the platelet glycoprotem Ia-IIa complex which can act as a divalent cation-dependent collagen receptor on nonacUvated platelets [139,206,207,219] Studies of the purified a 2 f l I receptor inserted into hposomes show magnesium dependence, with lnlublUon by high level of calcmm It binds to collagen types I, II, III and IV, but not to type V or gelatin According to a

104 h p o s o m e assay, there is no a d h e s i o n to flbronectln, lamlnln, or a variety of other p r o t e i n s The a subunit of this collagen receptor ( a 2 ) has been cloned a n d sequenced from h u m a n flbroblast c D N A (Ref 224, T a b l e III) It has an overall a m i n o acid sequence i d e n t i t y of 18 25% c o m p a r e d with other integrin a chains It contams most of the same cystelne residues, three p r e d i c t e d divalent c a t i o n - b i n d i n g d o m a i n s , a t r a n s m e m b r a n e dom a m , and a short c y t o p l a s m i c d o m a i n Recently, the a l f l 1 receptor has also been shown to b r a d to collagen [137], suggesting that some cells m a y be able to use several lntegrlns for interaction with collagen, ~ e cqfl 1, a 2 f l 1, and ot3/~1 A 90 k D a c o l l a g e n - b i n d i n g p r o t e i n t e r m e d ' C R I I I ' has also been identified b y C a r t e r a n d W a y n e r [33] C R I I I contains 25% c a r b o h y d r a t e and a 65 k D a p o l y p e p t i d e chain consisting of three structural d o m a i n s an external glycosylated d o m a i n , a h y d r o p h o b l c transm e m b r a n e d o m a i n , a n d an intracelhalar d o m a i n that contains a p h o s p h o s e r i n e It a p p e a r s to be a t r a n s m e m b r a n e p r o t e i n often co-locahzlng with v l m e n t l n C R I I I is a leading c a n d i d a t e for a possible t r a n s m e m b r a n e link between collagen and a specific cytoskeletal p r o teln The relationship between this p r o t e i n a n d the 88 k D a glycoprotein IV of platelets is unclear, G P IV IS a novel p r o t e i n that has recently r e p o r t e d to be a p r i m a r y receptor for collagen on platelets [229], a n d is also thought to be a t h r o m b o s p o n d l n receptor [13] Three c o l l a g e n - b i n d i n g p r o t e i n s from o s t e o s a r c o m a cells w~th m o l e c u l a r masses of 250, 70 a n d 30 k D a have been l d e n n f i e d using affinity c h r o m a t o g r a p h y on a collagen-hke triple helical synthetic p e p t i d e [54] T h e p e p t l d e was designed to c o n t a i n the R G D sequence a n d the purified p r o t e i n s were eluted with soluble R G D containing p e p t l d e s W h e n d e t e r g e n t - s o l u b i h z e d , these proteins recognize the R G D S sequence, but after reconstltUtlOn Into hposomes, they b i n d specifically to substrates p r e p a r e d with native collagen b u t n o t to substrates p r e p a r e d with d e n a t u r e d collagen, fibronectin, or vltronectm The relatlonshaps of the 70 a n d 30 k D a proteins to the 70 k D a p r o t e i n r e p o r t e d b y Sugrue [220] and anchorin CII, respectively, are not yet k n o w n

V. Laminin receptors Larmnin is a m u l t i m e r l c adhesive g l y c o p r o t e l n f o u n d mostly in b a s e m e n t m e m b r a n e s [150,233] It is a very large, c r o s s - s h a p e d p r o t e i n with one long a r m a n d three short arms (see Fig. 8) with a m o l e c u l a r m a s s of 900 k D a , of which 13% is c a r b o h y d r a t e It is c o m p o s e d of three d i s u l f i d e - b o n d e d p o l y p e p t i d e chains - one a chain of 400 kDa, one 225 k D a / 3 1 chain, a n d one 205 k D a fi2 chain The two fi chains are h o m o l o g o u s b u t n o t identical Each chain forms a different short arm, a n d all three chains together form the long a r m Like flbronec-

Tumor cell

--Collagen Hepann

-

-

Major hepann Neunte extension

Fig 8 Model of the structure of larmnm Lammm has a cross-shaped structure consisting of three polypeptlde chains termed a, /31 and/32 These chains mdwldually form each short arm of the cross, and all three together form the tong arm Larmnin contains binding regions for collagen type IV (stippled) and heparln (cross-hatched), and at least three d~stinct regions that interact with cells - a tumor cell adhesion region near the intersection of the arms (black), a hepatocyte attachment region at the top of the a chain short arm, and a neunte extension region at the end of the long arm (hatched)

tin, l a m l n l n a p p e a r s to be c o m p o s e d of protelnase-reSlstant d o m a m s , a n d the various b i n d i n g sites of l a m i n l n have b e e n m a p p e d (see F i g 8) L a r m n l n is a m a j o r c o n s t i t u e n t of b a s e m e n t memb r a n e s It was first isolated from the E H S mouse s a r c o m a [234], b u t has since been isolated from m a n y sources i n c l u d i n g c u l t u r e d cells [46], rat yolk sac t u m o r [65], a n d h u m a n p l a c e n t a a n d b a s e m e n t m e m b r a n e s as f r a g m e n t s [196] T h e a m o u n t of lan~nln can be reduced on the surfaces of t r a n s f o r m e d cells in culture [101] L a r m n i n was originally thought to be a specific adhesion p r o t e i n for e p l t h e h a l cells However, fibroblasts a n d s a r c o m a cells can also a t t a c h to l a m i n i n substrates [52,661. Cells c a n i n t e r a c t with larmnin through m u l t i p l e rec e p t o r systems (e g , see Refs 14 a n d 85) T h e 140 k D a avian r e c e p t o r c o m p l e x orig3nally identified as a f l b r o n e c t i n r e c e p t o r (but p r o b a b l y a mixture of fi~ integrms), c a n also interact with l a m m i n , although with low affinity [112] This 140 k D a receptor can also c o - l o c a h z e with l a m l n i n In vivo [63,138] F u r t h e r m o r e , a n n b o d l e s that recognize the 140 k D a r e c e p t o r system inhabit the a t t a c h m e n t a n d s p r e a d i n g of chicken and h u m a n cells on l a m l n l n substrates [50,112,236], as does a m o n o c l o n a l a n t i b o d y against the h u m a n fil s u b u n l t

[8]

105 Members of the Integnn fla family can function as lamlmn receptors for mammalian cells also. As noted above, aafl 1 can bind to laminln, along with fibronectln and collagen, ct6fl: from h u m a n platelets has been shown to be another lamlmn receptor, but with much more hgand specificity [217] Other laminln receptor integrins isolated from rat Rugh cells [76], and neuronal cells [122,236] are also fla mtegrlns, probably the homologues of a3fla or a6fl: The final placement of these larmnln receptors within the V L A / m t e g r l n classification system has yet to be completely resolved Lamlnln binds with high affimty ( K d = 2 - 4 10 -9 M) to 'receptors' (binding proteins) apparently consistmg of a single 67-70 k D a glycoproteln Lamlmn bmdmg proteins of this size have been isolated from human breast epithelial carcinoma cells [231], neutrophils [248], murine macrophages [115], fibrosarcoma cells [153], melanoma cells [195], rat myoblasts [147] and human placenta [241] Depending on the cell type, there are 10000-110000 receptors per cell [153,195,231] This 69 k D a laminm-blndlng protein has higher affinity than the fibronectin or v:tronectln receptors When transferred to nitrocellulose, it still binds laminln with relatively high affinity [58,147,153] Identification of the 69 k D a lammin binding protein as a true cell surface lamlmn receptor is somewhat controversial However, anti-69 k D a antibodies inhibit cell attachment and migration on laminln, while having no effect on fibronectin [242], and the 69 k D a protein has been localized on the cell surface [247] The lnteracnon of laminin with the 69 k D a binding protein reportedly depends on a short sequence of amino acids on the fll chain [86,87] Synthetic peptldes containing the sequence Tyr-Ile-Gly-Ser-Arg (YIGSR) can inhibit cell attachment to lamamn substrates, promote cell attachment when coated on plastic, and elute the 69 k D a protein from lamlnln affinity columns [86,87] A potentially related 69 k D a receptor that binds to elastm is a galactoslde lectln, and galactose can inhibit its function by releasing it from the cell surface [107] The relationship of this protein to the putative 69 k D a lamlnIn receptor remains to be determined The 69 k D a larmnln-blndlng protein can also bind to actin with moderate affmlty ( K 0 = 6 • 10 -7 M), perhaps along the sides of actin filaments, and promote the formation of actln bundles [24] These results originally suggested that this relatively small, monomeric receptorlike protein might span the plasma membrane, possibly inducing the organization of actln filaments by clustering at high receptor occupancy levels [49] On cultured endothelial cells, the localization of the 69 kDa lamlnin-blndlng protein varies, depending on the rmgratory state of the cell [247] Stationary cells have apical bmding protein localization in large orcular patches On migrating cells, lamlnin binding protein is restricted to ruffhng membranes (lamelhpodla) The

binding protein co-distributes with lammln on the exterior of cells and with actln filaments on the lntenor, again suggesting a transmembrane actln-lammIn link Confluent cells have actln in dense peripheral bands and in stress fibers with receptor in a diffuse pattern, co-distributing with the dense peripheral bands The 69 k D a larmnin binding protein from human colon carcinomas has been cloned and sequenced at the D N A level [241,249] Its sequence is surprising with amino acid residues accounting for only 33 kDa of the 69 k D a total mass There are no potential asparaginelinked glycosylatIon sites, although there are 24 potential O-hnked glycosylatlon sites on threonme and 12 on serine, enough to possibly account for the discrepancy in the molecular mass Although the receptor is composed of mostly hydrophoblc amino acids, there IS no leader signal sequence for transport into the ER and no obvious consensus membrane-spanning (transmembrane) region Unravehng its biochemistry and function will continue to be of considerable interest Neuronal cells may have multiple, independent larmnin receptors In addition to the 69 kDa lamlnin binding protein, they can also have a higher affinity 180 k D a receptor [132] or a 120-140 k D a binding protein [58]. The 180 k D a receptor is functionally and structurally distinct from the 69 k D a receptor and also the mtegnn receptors, and appears to be required for the extension of processes using the neunte-promoting site of larmmn To date, the 180 kDa receptor has been found only on neuronal cells The relationship of neurons to lamlnin is further complicated by the identification of a novel lammin-hke adhesive protein termed s-larmnln, which is also a potent promoter of neunte outgrowth [119] Relative affinmes for the binding of different lanunlns to the different laminin binding proteins can now be evaluated Another lamlnln receptor with a molecular mass of 110-120 k D a has also been described [132,214] This receptor has a relatively high affinity for lamlnln, binding well even after solubihzatlon in SDS and blotting to nitrocellulose [132,214] It ~s an integral membrane glycoproteln found on epithehal cells, a variety of transformed cells, and in tissues [132] Interestingly, despite ItS high apparent affinity to lamanln, it has not been observed as a larmmn binding protein under the conditions used for affinity purification of the 69 k D a bindlng protein. It wall be important to determine if this is a cell-type specific difference or a result of different techniques of detection A major lanunln-blndlng protein of 56 k D a characteristic of muscle has been isolated and cloned from chick and rat skeletal muscle [48,97]. This extracellular, membrane protein interacts with the major heparin-blndIng domain m the lammln a-chain The 406 amino acid protein contains a probable signal sequence and a site for N-linked glycosylataon, but no predicted trans-

106 membrane sequence The C-ternunus contains a peculiar repeat of 33 consecutive aspartate residues, and has been consequently named aspartactln [48]

VI. Conclusions The study of receptors for extracellular matrix proteins has advanced considerably over the past 2 - 3 years Most of the work to date has focused on the isolation of these receptors and the characterization of their structures Less is known about how these receptors regulate their functions Work on functional aspects has naturally emphasized the interactions of these receptors with extracellular molecules How these transmembrane glycoproteIns interact with antracellular proteins is of equal Importance and needs to be pursued vigorously Only the avian lntegrin complex has been determined to brad directly an Intracellular molecule (talln) Unfortunately, further work on the avian system has been severely hampered by its low binding affinity for fibronectln The development of blochermcal purification techniques for the receptor [130] may open the door to better functional assays for the avmn receptor Whether various integrin molecules function solely as physical links between extracellular molecules and cells, or whether they also have chemical signal transduction actiwties remains uncertain Their roles in cytoskeletal organizauon, m aspects of phagocytosls [88], m growth [237]. and conceivably even in proteanase regulation [240], need elucidation Functions in these and other complex processes such as cell m~gratlon will require clarification at the molecular level The techmques of molecular biology have provided crucial structural information However, the presence of putative calcium binding and glycosylation sites does not mean that they necessarily exist on the actual protern It will be important to determine directly the stoichiometry of calcium binding, precise numbers and structures of carbohydrates, and the presence and functions of other forms of covalent modifications, l e, acylatlon, phosphorylatlon, sulfatiom or O-linked glycosylatlon Another important question centers around the role of glycosylatlon of the mtegrln receptors This addresses the more general question of the role of glycosylation on receptors in general Because of the great general interest in the integrlns, they may provide valuable models for the study of the functional and structural roles of ollgosacchandes on glycoprotems The apparent anomalies in the structure of the 69 kDa lamanln binding protein as deduced from the c D N A sequence must also be explained Although early studies on the purified receptor suggested an integral membrane protein, the sequence data now indicate this is uncertain The possiblhty of an alternative membrane

anchor for ttus receptor is exciting but not known It~ relationship to the lectin-hke elastln receptor will also be interesting to unravel, as will the relative importance ol this protein versus the various lamlnln-bmdlng lntegrln receptors m different cellular processes The regulation of the extracellular matrix protein receptors as an important area of m~estigatmon The expression of these receptors in wvo should be examined to compare with current m vitro models to avoid artefacts It will be important to compare how these receptors are regulated during wound healing, metastasis and development In addition, the existence of multiple putative receptors for each extracellular adhesive protein raises the posslblhD of different receptor recognition sates on each protein, and consequently of possible differential receptor regulatIon during development or other processes These studies wall require antibodies speclflC for each receptor, e g, for each lntegrln a-subunlt Besides descriptive analyses, such antabo&es can be used for Inhibition studies to examine the role of each receptor m VlVO Complementary inhibition or expression experiments using naturally occurring mutations and molecular biology approaches will also be needed At least one Drosophtla lntegrm mutant is known, which defines a role for this molecule surprisingly late in development, however, the hgand of this particular integrin is unknown, and may be a musclespecific type of integrin molecule [17,146,152] Possible tissue-type specificity of integrlns reqmres further m,,est~gatlon To test the function of vertebrate lntegrlns, the most fruitful approaches may involve inhibition using anti-sense D N A and expression using cloned receptor c D N A s and mutants expressed artxflcmlly m novel locations or at different times All of these studies should help define the roles of these molecules in ~avo In general, the broad distribution and wide-ranging potential functions of these extracellular matrix protein receptors will make them very interesting, important, and productive subjects of research for the foreseeable future

Acknowledgments S K A was partially supported by grants CA-14718 and CA-45515 awarded by the National Institutes of Health, U S P H S, D H H S

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Cell surface receptors for extracellular matrix components.

91 Btochlmtca et Blophystca Acta, 1031 (1990) 91-110 Elsevier BBAREV 85361 Cell surface receptors for extracellular matrix components Steven K. Akiy...
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