J. ADAMS AND J. LAWLER EXTRACELLULAR MATRIX

The thrombospondin family Extracellular matrix molecules that are related to the

adhesive glycoprotein thrombospondin and expressed in a variety of different tissues have recently been identified.

Recent years have witnessed a new appreciation of the roles of extracellular matrix glycoproteins, such as fibronectin and laminin, in regulating cell behavior, prolif eration and differentiation. This has come about, in part, through an improved understanding of the molecular mechanisms by which these glycoproteins interact with cells. Meanwhile, the possible functions of other matrix molecules remain less well understood. Thrombospondin is a glycoprotein of the extracellular matrix that was first isolated as a 420 k~, trimeric molecule released from the u-granules of thrombin-stimulated platelets. It has since become clear that thrombospondin is present in the extracellular matrix of developing and repairing tissue and that it has adhesive and growth-promoting activi- ties in vitro Taken together, these properties su%cst that thrombospondin may be a unique matrix compo- nent that facilitates cell movement. One focus of current research is the identification of cellular binding sites and specific receptors for thrombospondin [ 1,2]. The recent discovery of a family of thrombospondin-related pro- teins has added impetus to this endeavour, and forces a reappraisal of the roles attributed to thrombospondin in the the extracellular matrix.

Molecular cloning of the first identilied form of throm- bospondin (now called TSP-1) revealed that each subunit of the trimer consists of multiple domains: amino- :;ad carboxy-terminal globular domains, a region of sequei EX similarity to procollagen, and three types of repeated XI- quence motifs, designated type 1, type 2 and type 3 re- peats (Fig. 1). The type 1 repeats have attachment sites for various cell types, type 2 repeats resemble repeats in epidermal growth factor (EGF) and type 3 repeats form a series of Ca2+-binding loops. !A the past year, four proteins have been identified that are structural?~; related to TSP-1, all of which are products of separa;?: genes- TSP.2 [3,4], TSP3 [5], TSP.4 [6] and COW (cartilage oligomeric matrix protein) [7]. These proteins fall into two subgroups, as TSP-1 and TSP-2 (subgroup A) have the same set of structural domains, whereas TSP3, TSP4 and COMP (subgroup B) have non-related amino- terminal domains of different sizes, do not have regions related to procollagen or type 1 repeats, and do contain a fourth type 2 repeat (Fig. 1). The sequence relationships between these Ike molecules suggest that the thrombospondin family arose through a

Subgroup A TSP-1

TS P-2

TSP-3

Subgroup B TSP-4

COMP

67 Amino-terminal domains Pair of conserved

cysteine residues

Procollagen similarity Type 1 repeats

Type 2 repeats Type 3 repeats

Carboxy-terminal domains

Fig. 1. Structures of thrombospondin family members. When compared with TSP-1, the newly identified proteins have a gradient of sequence identity from amino to carboxyl termini, ranging from 30 to over 80% identity for TSP-2 and from about 12 to 60% for sub- group B members; darker colours indi- cate greater sequence similarity. Sub- group B members have a 15 amino-acid extension at their carboxyl termini.

@J Current Biology 1993, Voi 3 No 3

series of gene duplication events, the f&t of which oc- curred approximately 900 million years ago and gave rise to the progenitors of the two subgroups [8]. More recent duplications have then given rise to the individual forms observed in vertebrates today. On this evolutionary time scale, it would be predicted that an invertebrate form of thrombospondin did exist at one time, although none has yet been identified in modern invertebrates. Many vertebrate and invertebrate proteins do, however, contain segments with sequences related to the individ- ual domains of thrombospondin-for example, domains similar to the amino-terminal globular domain of TSP-1 are present in vertebrate collagens type V, IX, XI and xu [9]. Other proteins of the extracellular matrix, such as tenascin, laminin and fibrillin, contain EGF-like repeats, and this type of repeat is also found in many other proteins of both vertebrates and invertebrates. Finally, domains similar to type 1 repeats, also sometimes called ‘thrombospondin repeats’, are found in a growing list of proteins that are involved in adhesive processes. These include late components of the complement cascade that is involved in antibody-mediated cell lysis, coat proteins of the blood-borne and circumsporozoite life stages of malarial parasites [a], and me proteins F%pondin [lo] and UK-5 [ 111, which are involved in axpn guidance. As type 1 repeats are only present in TSP-1 and TSP-2, it is likely that the exons encoding them were ‘shuffled’ in to the progenitor of subgroup A after the original gene duplication event. With the identification of several new thrombospondins, it has now become cltrdr that the ha& mark of the thrombospondin family is, in fact, the pres- ence of the type 3 repeats and carboxyterminal domain, which are the most conserved in sequence between the &e family members. What clues are there regarding the functions of the newly described tbrombospondins and how they might diKer from those of TSP-l? Even with the limited amount of information currently available, it is clear that individual thrombospondiis are expressed in quite tierent tis- sues and are present post-natally as well as in embryos. Transcripts encoding TSP.1 and TSP-2 are found in a

wide range of tissues, with the refauve levels of expres_ sion of the two differing between tissues [4], m avian embryos, TSP-2 is most highly expressed by connec tive tissue, epimysium and endothelial cells, and cannOt be detected in the developing nervous system by i?~ situ hybridization [12]. By contrast, TSP-3, TSP-4 and Coup each have much more restricted distributions: TSP 3 is most highly expressed in lung and skin 151, TSP-4 in heart and skeletal muscle [6], and COMP only in cam lage, after the initial stage of chondrogenesis [ 131. These data suggest that a set of thrombospondins, including representatives of both subgroups, forms part of many ex- tracellular matrices, and raise the possibility that still more tissue-specific thrombospondins remain to be discovered. The presence in the precursors of the four newly de- scribed thrombospondins, of ‘signal sequences’ respon- sible for targeting proteins for secretion, indicates that, like TSP-I, they function extracellularly. As the two cys- teine residues responsible for trimerization of TSP.1 are also present in the other family members, it is likely that all are multimeric. TSP-2, like TSP-1, forms trimers [14], but COMP is secreted as a pentamer [15]. No reagents are yet available to track possible multimers of TSP.3 and TSP-4. Although the simplest model is that each throm- bospondin forms a homomer, the observation that TSP-1 and TSP.2 are also capable of forming heterotrimers [ 141 suggests that additional levels of complexity are possible. Indeed, if heteromers also form between members of the two subgroups, the number of potential thrombospondin multimers could be in the hundreds. Many of the molecular functions of TSP.1 have now been mapped to particular domains of the molecule (Table l), allowing predictions of the functions of the other fam- ily members to be made. Clearly, TSP.2 is most likely to have similar activities to TSP-1, but functions that map to me carboxy-terminal half of TSP-1, such as Cazf-bmd- ing and cell adhesiveness, may well be shared by mem- bers of both subgroups. A series of cell-binding sites and thrombospondinbinding receptors have now been identified for TSP-1, although individual sites have yet to be conclusively linked to particular functions of TSP-1.

Table 1. Map of the functional domains of the TSP-1 molecule.

Receptors bound Syndecan, heparan sulphate proteoglycans, sulphatide.

neurite outgrowth.

I platelet aggregation.

I I

190 Current Biology 1993, Vol 3 No 3

Comparison of the protein sequences cor&rns that the new thrombospondins may interact with ceils in ways similar to TSP-1. Two of the type 1 repeats of TSP-2 have the amino-acid sequence CSVTCG, which binds to heparin and mediates adhesive interactions with many cell types in vitro. This motif may bind to several types of cell surface receptors (Table 1). TSP-2 also has the sequence RGD, known to bind to integrins, within its last type 3 repeat - like TSP-1, TSP-2 may bind to @, integrins. Melanoma ceils attach to the sequence motif PYVVMWK in the carboxy-tenninaI globular domain of TSP-1 [16], and this motif is conserved in all five family members [4-71. The nature of the receptor for this sequence on melanoma celis is unknown at present, although candi- date receptors for the carboxyterminal domain of TSP-1 have been identified in other cell types (Table 1).

Some features of the newly described thrombospondins suggest that they may also interact with cells in ways distinct from TSP-1. For example, the amino-terminal heparin-binding sequences of TSP-1 are not completely conserved in TSP-2, and TSP-2 binds heparin with lower afEn@ than TSP-1. Thus, in tissues where TSP-2 predom- mates over TSP-1, interactions with cell surface heparan sulphate proteogiycans may be modulated [14]. In ad- dition, the ammo-terminal globular domains of the sub- group B proteins are not related to those of subgroup A, and do not contain hepann-binding sequence motifs [5-71; these may turn out to encode unique or tissue- specific functions. Although the RGD integrin-binding se quence of the last type 3 repeat is conserved in TSP-1 from humans to frogs [ 8 1, there is no RGD sequence at this site in thrombosponciins of subgroup B [5-71. The adhesive sequence IRWM in the carboxy-terminal domain of TSP- 1 [ 161 is also not present in any of the newly described proteins. As the heparin-binding and carboxyterminal do- mains of TSP-1 mediate many of its activities [l], the new family members may prove to have quite different effects on celi behavior. The subgroup B proteins also lack type 1 repeats and so may lack the neurite outgrowth-promoting activities of TSP-1; they may also interact differently with other matrix components [2].

It is generally thought that the existence of separate genes encoding functionally related proteins may serve to in crease the possibilities for transcriptional regulation, and TSP-1 and TSP-2 mRNA levels do indeed change in re- sponse to diiferent stimuii [2] I However, as subgroup B members may have unique functions, the expression in individual tissues of an array of thrombospondins may indicate that normal tissue function requires the fuil range of functions provided by the two subgroups. At the molec- ular level, the different members of the thrombospondin family may prove to interact with both common and unique receptors. In this respect, it is tantalizing that there is very little information available on potential re- ceptors for the carboxytenninai domain, even of TSP-1, although it is the region of highest sequence conservation between family members. Perhaps a whole set of throm- bospondin receptors remain to be discovered. The tissue- spectic expression patterns of TSP3, TSP.4 and COMP

suggest appropriate cell types in which to address these questions. With all this on the agenda, thrombospondins are likely to be intensely studied for some time to come,

Acknodedgements We thank V Dkit and R Tucker for commute+ tion of data prior to publication.

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Josephine Adams and Jack Lawler, Vascular Research Di- vision, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue Boston, Massachusetts 02115 USA