692

ADAMs and cell fusion Ari-Pekka J Huovila*t, Eduardo AC Almeida* and Judith M White*
Members of the ADAM family (membrane proteins with a disintegrin and metalloprotease domain) have been implicated in several cell-interactive events, including cell-cell fusion. Recent evidence implicates three ADAMs, fertilin-~, fertilin-13, and meltrin-eq in sperm-egg fusion and myoblast fusion. In light of the large number and wide tissue distribution of the ADAMs, they may also participate in other cell-cell fusion events. As ADAMs are also found in both vertebrates and invertebrates, some features of cell-cell fusion reactions may be conserved throughout the animal kingdom. Table 1
Potential functions of ADAM ectodomalns.
ADAM ADAM 2 Synonyms Metallopretease? * Adhesion? ** * ** ** ** * ** * * ** Fusion?

ADAM 1

Fertilin-e
Fertilin-I~

ADAM 3 ADAM 4 ADAM 5 ADAM 6 ADAM 7 ADAM s ADAM 9

ADAM ADAM ADAM ADAM ADAM 10 11 12 13 15

Cyritestin, tMDC I
tMDC II

tMDC IV EAP I MS2 MDC 9

MP cDNA MDC Meltrin-a Metargidin, MDC 15, vascular MDC

Addresses *Department of Cell Biology, University of Virginia, Health Sciences Center, PO Box 439, Charlottesville, VA 22908, USA tDepartment of Neurology, University of Kuopio, PO Box 1627, RN-70211 Kuopio, Finland Se-mail: jwTg@virginia.edu Correspondence: Judith M White Current Opinion in Cell Biology 1996, 8:692-699

Current Biology Ltd ISSN 0955-0674

Abbreviations ADAM protein containing a disintegrin and metalloprotease domain SVMP snake venom metalloprotease

An asterisk in the 'metalloprotease' column indicates that the specified ADAM contains the consensus metailopretease site amino acid sequence (the HEXXH box). All ADAMS have been given at least one asterisk in the 'adhesion' column as they all contain a predicted disintegrin loop. Those ADAMs whose predicted disintegrin loop 'active site' tripeptide contains a negative charge in the third position which is conserved in all the cress-species homologs are indicated with a double asterisk. One asterisk in the 'fusion' column indicates that the specified ADAM contains a predicted fusion peptide. References for ADAMs 1-11 are given in [3 '] and those for ADAMs 12 and 15 are given in [23"'] and [43], respectively. The gene encoding ADAM 13 was cloned from Xenopus/aevis (D Alfandari, TG Wolfsberg, JM White, D DeSimone, unpublished data). EAP, epididymal apical protein; MDC, metalloproteinase-like, disintegrin-like, cysteine-rich protein.

Introduction
Membrane fusion events are important both intracellularly, for example in vesicular trafficking [1'], and on the cell surface, for example in virus-cell and cell-cell fusion [2]. In this review, we focus on cell-cell fusion and, in particular, on recent evidence implicating members of a newly discovered gene family, the ADAMs (proteins containing a disintegrin and metalloprotease domain) ([3",4"]; see Table 1), in two important cell--cell fusion events, sperm-egg fusion and myoblast fusion. Cell-cell fusion events are critical to the development of multicellular organisms. For example, fusion of sperm and egg initiates zygotic development; fusion of myoblasts generates large contractile units called myotubes; fusion of certain monocytes generates osteoclasts which are boneresorbing cells; and fusion of cytotrophoblasts generates a syncytial lining of the placenta. Before two cells can fuse, however, they must attain fusion competence and this involves a series of steps as outlined in Figure 1. First, the cells must differentiate, a process that may involve changes in gene and protein expression, proteolytic processing of cell surface proteins, and changes in cell shape (Fig. la). Second, the cells must recognize and adhere to their fusion partner (Fig. lb). In some cases, notably in sperm-egg fusion, this involves prior cell migration. Lastly, the cells must fuse, that is they must merge their separate bilayers into one (Fig. lc). After fusion has occurred,

new developmental programs are initiated in a manner that probably involves fusion-mediated signal transduction (Fig. ld).

Figure 1

=~>

I (a)

I (b)

I I (c)

II (d)

1g96 Current Opinion in Cell Biology

Steps in cell-cell fusion. In order to fuse, cells must: (a) differentiate so as to attain fusion competence; (b) adhere to one another; and finally (c) fuse (i.e. merge their outer lipid bilayers). After fusion, there are likely postfusion signaling events (d) involving changes in cell morphology and physiology. This figure does not show either heterotypic or homotypic fusion in particular; the fusing cells are colored differently only to differentiate them from each other.

Two principles have guided our work on cell-cell fusion. T h e first is that, like all biological membrane fusion reactions, cell-cell fusion events are both energetically unfavorable and specific [2,5]; they will therefore require

ADAMs end cell fusion Huovila, Almeida and White 693

proteins in order to become energetically favorable and specific. T h e second is that as cell--cell fusion events are topologically equivalent to virus--cell fusion events, the proteins that mediate cell-cell fusion reactions will share traits with viral adhesion/fusion proteins [2].
A D A M s in s p e r m - e g g fusion Identification of the ADAMs

has the sequence QDEC (single-letter code for amino acids) at the corresponding position. As discussed below, recent evidence has implicated the predicted fertilin-13 disintegrin loop in sperm-egg binding. As fertilin-(x shares properties, most notably a putative fusion peptide, with viral fusion proteins, it is considered to be a candidate fusogenic subunit. Viral fusion peptides are short stretches of mostly hydrophobic amino acids found in a transmembrane subunit of viral fusion protein complexes. Most viral fusion peptides show a tendency to form an amphipathic ot helix and 13 sheet [2]. T h e candidate fusion peptide in guinea pig fertilin-( shares these characteristics as well as limited sequence relationship with an apolar sequence found in the rubella virus E2 glycoprotein [7]. Another prominent feature of viral fusion proteins is that they are oligomeric. T h e fertilin complex on the sperm plasma membrane consists of a tight complex of two transmembrane glycoprotein subunits [13], and these appear to be further oligomerized (SI Waters, JM White, unpublished data). In further analogy with viral fusion glycoproteins, the fertilin subunits are proteolytically processed from larger precursors [13]. Interestingly, the final proteolytic processing event that has been detected in fertilin-13 takes place coincident with the acquisition of fertilization competence; this phenomenon is similar to the activation of the fusogenic potential of viral fusion proteins by proteolytic cleavage.
Model for fertilin-mediated membrane fusion

A search for proteins involved in sperm-egg fusion that share rudimentary traits with viral adhesion/fusion proteins led to the characterization of fertilin [3",4",5]. Fertilin is a heterodimer found on the sperm surface and is composed of two type I integral membrane glycoproteins, namely fertilin-(x and fertilin-13, the prototypes of the ADAM family. Fertilin was originally implicated in sperm-egg fusion on the basis of the observation that an anti-fertilin monoclonal antibody inhibits fusion in vitro [6]. T h e cloning of cDNAs encoding the fertilin subunits as apparently expressed on mature guinea pig sperm revealed the presence of a putative disintegrin domain in the I 3 subunit and a putative fusion peptide in the a subunit [7]. This led to the hypothesis that fertilin binds to an integrin on the egg surface via the disintegrin domain of its 13 subunit, and that the (x subunit then mediates the union of the opposed plasma membranes.
Comparison of ADAMs with viral fusion proteins

The hypothesis that fertilin may be involved in fusion (Fig. lc shows fusion) is based mainly on a comparison of fertilin with viral fusion proteins. Fertilin displays most of the characteristics of viral adhesion/fusion proteins [2], most notably a binding domain in the fertilin-13 subunit and a possible fusogenic motif in the fertilin-a subunit. The binding domain in the fertilin -13 subunit is its 'disintegrin' domain (Fig. 2). Disintegrins were originally isolated from viper venoms as short soluble polypeptides that disrupt integrin-mediated cell-cell and cell-matrix interactions, hence the name disintegrins [8]. Soluble disintegrins are proteolytically formed from larger precursors known as snake venom metalloproteases (SVMPs) [9,10]. All three subclasses of SVMPs, namely PI, PII and Pill, contain a zinc-dependent metalloprotease domain; Pill SVMPs also contain a further downstream cysteine-rich domain. T h e disintegrin domains of SVMPs bind integrins with high affinity and inhibit integrin-mediated platelet aggregation and cell-matrix attachment, thus allowing spread of the venom and promoting hemorrhage in the bite victim. As fertilin-13 is a transmembrane protein, however, its disintegrin domain is thought to mediate a direct cell-cell interaction via binding to an integrin. PII disintegrins display a 13 amino acid projecting loop with sequences such as KGD, MVD, and RGD (single-letter code for amino acids) at the tip of the loop [11,12]. Pill disintegrins are instead predicted to have a 14 amino acid loop with sequences such as ESEC (single-letter code for amino acids) at their predicted tips. As shown in Figure 2, the predicted disintegrin loop of mouse fertilin-13

A working model for fertilin-mediated membrane fusion depicts an initial binding interaction between fertilin-13 and a receptor on the egg plasma membrane. It is further proposed that following binding there is a conformational change in fertilin that exposes the candidate fusion peptide in fertilin-(x, thereby allowing it to bind hydrophobically to the egg plasma membrane [14]. By analogy with viral models [15], the next steps of fusion are envisioned to involve clustering of several fertilin complexes at the fusion site, mixing of the outer leaflets of the lipid bilayers, hemifusion and, subsequently, full fusion involving the opening and dilation of a fusion pore [15]. T h e fusion complex on the sperm membrane could consist of fertilin subunits alone, or it could contain other accessory proteins. Alternatively, instead of fertilin-(~, another protein might mediate the actual membrane fusion reaction. T h e first part of the hypothesis for fertilin-mediated binding and fusion of sperm and egg is supported by two lines of evidence. T h e first is that an integrin ((x6131) on the surface of unfertilized mouse eggs has been identified as a key participant in sperm-egg binding [16"']. T h e second is that peptide analogs of the fertilin-I~ disintegrin domain inhibit sperm binding to eggs or to somatic cells that express the (x6131integrin [ 16, 17,18"]. Integrins have also been implicated in the fertilization of human and hamster eggs [19,20].

694

Cell-to-cell contact and extracellular matrix

Figure 2 Domain structure of ADAMs. Full-length ADAM precursors contain a signal sequence (ss), followed by a prodomain (P), a metalloprotease-like domain (M), a disintegrin domain (D), a cysteine-rich region (C), a transmembrane domain (tm), and a cytoplasmic tail (t). The amino acid sequences of the metalloprotease active site regions and the disintegrin loops are shown for mouse fertilin-(x and -13 and meltrin-(x. The candidate fusion peptide regions (in the C region) are shown for mouse fertilin-ot and meltrin-(~. The amino acids at the tip of the predicted disintegrin loops are denoted by asterisks. The putative fusion peptides are indicated by thin horizontal lines. The arrow indicates the position of the proteolytic processing step that is postulated to generate the 'mature' fertilin-a and fertilin-13 and 'putative mature' meltrin-c~ subunits. Fusion peptide region sequences are aligned together as presented in [ 2 3 " ] ; the dashes in the fertilin sequence are gaps so as to allow for the best alignment over the entire C domain. The amino terminus is shown at the left of the figure.

ss

tm

II

III

Fertilin-13
Meltrin-(x

CRLAQDECDVTEYC
CRGSSNSCDLPEFC

F Fertilin-c(
Meltrin-c~

KLVCTDVRYL PKVK .... PLHSLLQVPYGE~C KI QCQGGASRPVIGTNAVS IETNI PQQEGGRIL

1996 Current Opinion in Cell Biology

Three lines of indirect evidence support the hypothesis that fertilin-~ serves a fusogenic role. First, cDNAs encoding fertilin-~ subunits that have been cloned to date, from several different species [3,7,21], contain a candidate fusion peptide that can be modeled as an amphipathic a helix or 13 sheet. Figure 3 shows hydrophobicity plots, in addition to a-helix and ~-sheet projections, of the putative fusion peptides of guinea pig and mouse fertilin-~. Second, to begin to test the possibility that the putative fusion peptide of fertilin-c~ can function as such, Muga et al. [22] synthesized a peptide corresponding to the putative fusogenic region of guinea pig fertilin-ct. T h e peptide was found to bind to phospholipid membranes and to induce fusion. Third, another ADAM with a putative fusion peptide, meltrin-a, was recently implicated in myoblast fusion [23]. However, despite much suggestive evidence, it is premature to conclude that fertilin-ct is a cellular fusion protein. T h e critical question is whether the candidate fusion peptide (Figs 2,3) truly functions as such. As has been the case for viral fusion peptides [24], answering this question will probably require a combination of transfection studies, site-specific mutagenesis, and biochemical and biophysical studies including labeling with photoactivatable phospholipid labels. Genetic knockout experiments may also aid the endeavor. If future work indicates that fertilin-~ is not involved in membrane fusion p e r se (fusion is shown in Fig. lc), then we would expect there to be another protein on the sperm (or egg) surface that fulfills the function of

membrane merger. This might be another ADAM, as other ADAMs are expressed in spermatogenic cells [3,25,26], or it might be another antigen implicated in sperm-egg fusion [27-29,30]. If fertilin-oc does not mediate fusion p e r se, it may have an earlier function in the pathway leading to sperm-egg fusion (Fig. 1; [14,28,30]). For example, the precursor region of fertilin-a contains a metalloprotease domain that is predicted to be catalytically active ([31]; Fig. 2). This metalloprotease domain could be involved in processing critical sperm antigens during sperm differentiation. Alternatively, fertilin-~ might facilitate sperm migration through the testis or release of sperm into the lumen of the seminiferous tubules. As fertilin-a on testicular sperm has been processed so that the metalloprotease domain is no longer covalently attached, we do not currently think that the metalloprotease domain is involved in sperm movement through the male or female reproductive tracts. It is also possible that the disintegrin domain of fertilin-~ may be involved in sperm-egg binding. Although mature fertilin-et as purified from guinea pig sperm did not possess a full disintegrin domain [7], purified mature bovine fertilin-a does (SI Waters, JM White, unpublished data).
General characteristics of t h e A D A M s

As mentioned above, fertilin-oc and fertilin-[3 are the prototypes of the ADAM gene family (see Table 1). Figure 2 shows the general domain structure of ADAMs, deduced from their cDNA sequences [3',4]. Newly

ADAMs and cell fusion Huovila, Almeida and White 695

Figure 3 The putative fusion peptides of guinea pig and mouse fertilin-(z and mouse meltrin-(x. (a) Kyte-Doolittle hydrophobicity plots are shown. Peaks above the x-axis are hydrophobic regions (e.g. the strongly hydrophobic transmembrane domains). The horizontal bars indicate the position of the candidate fusion peptides (fp). tm, transmembrane domain. The x-axis represents position along the amino acid sequence. The y-axis represents hydrophobicity value. (b) e.-helix projections of the ADAMs are shown; shaded areas indicate possible hydrophobic faces, and white letters indicate hydrophobic amino acids (hydrophobicity values >0.48 on the normalized consensus scale of Eisenberg [44]) within these areas. The average hydrophobicities of these faces are 1.3, 1.1, and 0.8 for guinea pig fertilin-cq mouse fertilin-(x, and mouse meltrin-cq respectively. (c) [~-sheet projections of the ADAMs are shown. Hydrophobic amino acids are highlighted. (a) tm i-5 0 ~ ~P"~ L3 ~tm r 4 M

'
,y'e,y',q , v . V ,~ ~ -

Guinea~pig fertillin-(~ fP

Mouse fertillin ot fP .... :,. ZJ~. Me~rtr:~ f ~ ~ffi~rv/w ~ V l ~ / ~

-tm

L-3 f4 ro ~ ~ / ~ t - 3

A . . . .

(b)

1;

Guinea pig fertillin-(~

Mouse fertillin-c~

Meltrin-c(

(e)

Guinea pig fertillin-c(

Mouse fertillin-(x
~ ~ ]i~i ! !~ ii

Meltrin-c~

synthesized full-length ADAMs contain a prodomain at their amino-terminal end, followed by a metalloproteaselike domain. Some ADAMs, notably fertilin-ot, have the consensus sequence for the active site of a zinc-dependent

metalloprotease ( H E X X H X X G X X H [single-letter code for amino acids]); this sequence is similar to those found in the active sites of SVMPs [3',4,31]. All ADAMs have a disintegrin domain that may bind integrins or other

696 Cell-to-cell contact end extracellular matrix

receptors [3"',4",31]. The critical amino acid residues at the tip of the putative integrin-ligand motif are indicated by asterisks in Figure 2. In some ADAMs, notably fertilin-(~, the cysteine-rich region following the disintegrin domain contains a relatively hydrophobic sequence that is similar to certain viral fusion peptides (Figs 2,3). All ADAM cDNAs encode a predicted membrane-spanning sequence, followed by a cytoplasmic tail of varying length. T h e predicted cytoplasmic tails of some ADAMs contain possible regulatory sites that may be involved in signaling ([32]; SI Waters, JM White, unpublished data).

ADAMs

and myoblast fusion

T h e formation of myotubes from myoblasts is, like fertilization, a complex muhistep process. Furthermore, like sperm, prefusion myoblasts must differentiate before they can fuse. Following differentiation, myoblasts align with, and adhere to, each other, and then merge their plasma membranes to form muhinucleated myotubes [33,34]. T h e process of myoblast adhesion (Fig. lb) is itself composed of a complex series of molecular interactions that have been classified as calcium-dependent or calcium-independent [34]. Several well characterized cell adhesion molecules (CAMs), including cadherins, neuronal and vascular endothelial CAMs, and integrins, have been implicated in myotube formation [34]. How myoblasts actually fuse and which cell surface proteins are critically involved in membrane merger remain, however, challenging problems. T h e most recent protein implicated in myoblast fusion is an ADAM called meltrin-(x [23"']. On the basis of the observation that two members of the ADAM family, fertilin-(~ and fertilin-13, are involved in sperm--egg fusion, Yagami-Hiromasa et al. [23"'] searched for fertilin homologs in muscle cells and cloned cDNAs encoding three proteins, mehrins (x, ~, and y. Of these, the expression of mRNA encoding meltrin-(x was found to be the most restricted; it is expressed mainly in skeletal muscle and, at a lower level, in bone. (The finding of meltrin-a in bone is potentially interesting because fusion of monocytes to form muhinucleated osteoclasts occurs in bone.) As further evidence for a role of meltrin-a in myotube formation, Yagami-Hiromasa et al. [23"'] showed that when C2 mouse myocytes are induced to differentiate, expression of meltrin-(x mRNA increases, with a similar time course to that of myogenin, an early marker of myoblast differentiation. Thus, they considered it possible that meltrin-(x participates in myoblast fusion. Like other ADAMs, the predicted full-length meltrin-(x contains a metalloprotease domain and a disintegrin domain. In addition, like fertilin-~, meltrin-ot contains a potential fusion peptide (Figs 2, 3) which shows limited sequence relationship to the fusion peptide of Sendai virus. T h e candidate fusion peptide of meltrin-~ is, however, less hydrophobic than the candidate fusion peptides of fertilin-(x (Fig. 3).

Yagami-Hiromasa et al. [23 "] next conducted a functional test of the role of meltrin-et in myoblast fusion. When differentiating C2 myocytes were transfected with full-length meltrin-(x cDNA, fusion was slowed. Conversely, when a shortened version of meltrin-a lacking the prodomain and metalloprotease domain (Fig. 2) was transfected into the same cells, fusion was accelerated and the fusion products were 'myosacs', round structures with irregularly distributed nuclei, instead of the characteristic elongated myotubes with aligned nuclei. Myosacs have previously been shown to form after the application of microtubuledisturbing agents and PMA (phorbol myristate acetate), a protein kinase C activator, to developing myoblasts. Meltrin-~ has a relatively large cytoplasmic tail that could be involved in interactions within the fusing cells, such as with cytoskeletal or signaling molecules. Hence it was suggested that one function of meltrin-et might be to help regulate myotube morphology [35]. T h e opposite effects of full-length meltrin-(~ and the construct lacking the prodomain and metalloprotease domain suggested that proteolytic processing may be important in regulating the function(s) of meltrin-(x. Yagami-Hiromasa et al. [23"'] showed that developing muscle cells contain both the 'full-length' version of meltrin-(x and smaller forms. T h e smaller forms could correspond to versions that lack, progressively, the prodomain and the metalloprotease domain. Yagami-Hiromasa et al. did not, however, evaluate which forms of meltrin-~ are expressed on the surface of developing myoblasts. By analogy to fertilin, the smallest form of meltrin-~ (58 kDa) could be the 'mature form' containing the disintegrin and downstream regions (Fig. 2, right of the arrow) and could, potentially, be involved in myoblast adhesion, fusion, or signaling. The largest form containing the upstream prodomain and metalloprotease domain could be a precursor in which the downstream functions of meltrin-(~ (e.g. proteolysis, adhesion, fusion, or signaling) are masked. Expression of the precursor might prevent myoblasts from anomalous premature fusion. An intermediate form in which the prodomain, but not the metalloprotease domain, has been removed could be involved in proteolytic events that are prerequisites to myoblast fusion. Yagami-Hiromasa et al. [23"'] speculate that meltrin-(x could be involved in degrading the extracellular matrix. Meltrin-a may also be involved in proteolytically activating itself or other myoblast surface proteins. In both viral fusion and sperm-egg fusion, proteolytic processing of surface antigens is essential for subsequent fusion [2]. Transfection of meltrin-~ into fibroblasts did not promote adhesion or fusion, suggesting that meltrin-e( alone does not function as a homotypic adhesion or fusion molecule [23"']. As the authors suggested, meltrin-(~ may exist as part of a heterodimer (or higher oligomer) as do fertilin-ot and fertilin-13, or it may need other factors to exert its function. T h e presence of a disintegrin-like

ADAMs and cell fusion Huovila, Almeida and White

697

domain suggests that meltrin-a may be involved in cell adhesion, which is conceivable as myoblasts express integrins on their plasma membrane [36,37]. T h e short form of meltrin-a [23 ] contains the entire disintegrin-like domain, but the sequence at the apex of the putative disintegrin loop (amino acid sequence SNSC; see Fig. 2) does not contain an acidic residue at the third position (as does, for example, the sequence Q D E C in mouse fertilin-~), a common feature of fertilin-13 and Pill snake venom hemorrhagic disintegrins. T h e sequence requirements for a functional disintegrin loop remain, however, to be determined for the cases of both integrin and potential nonintegrin [38] receptors. Thus, meltrin-~ could bind to an integrin or a nonintegrin receptor on its fusion partner. In summary, it has been demonstrated that meltrin-~ is involved in the pathway leading to myoblast fusion [23]. As is the case for fertilin, however, there is as yet no evidence implicating meltrin-c~ in membrane fusion per se. T h e possible roles of meltrin-ct in proteolysis, cell adhesion, cell fusion, and cell signaling must now be tested. Whether or not meltrin-~ is involved in fusion per se, it is likely that additional proteins, perhaps other ADAMs [3"], are needed to orchestrate the complex molecular interactions that lead to myoblast-myoblast fusion.

Given the large number of ADAMs and their wide distribution [3,4], it is likely that ADAMs participate in other cell--cell fusion events. In addition to fertilin-a and meltrin-cx (ADAMs 1 and 12), ADAMs 9 and 11 contain putative fusion peptides [3,4]. One ADAM, ADAM 8, was cloned from macrophages which form multinucleated giant cells during inflammatory reactions [39]. ADAM cDNAs are also expressed in the placenta, where cytotrophoblasts fuse to form syncytiotrophoblasts (GenBank access number H12627; PD Straight, JM White, unpublished data). Meltrin-ct is also expressed in bone, where monocytes fuse to form osteoclasts. ADAM cDNAs have been cloned from Xenopus (ADAM 13; D Alfandari, T G Wolfsberg, JM White, D DeSimone, unpublished data), Drosophila (GenBank access number G01204), and Caenorhabditis elegans (ADAM 14; B Podbilewicz, personal communication; see also [4]). T h e first ADAM found in C. elegans (ADAM 14) is present in ceils that undergo fusion reactions in the hypodermis ([40]; B Podbilewicz, personal communication). As ADAMs are found in lower animals in cells that undergo cell-cell fusion, it appears plausible that at least some features of cell-cell fusion reactions are conserved throughout the animal kingdom. There may, however, be important exceptions. Cell surface proteins that are unrelated to ADAMs have been implicated in sea urchin and Chlamydomonas fertilization [41,42]. ADAMs are also not likely to be involved in yeast mating, because the genome of Saccharomyces cerevisiae does not appear to contain ADAM genes (TG Wolfsberg, personal communication). Thus, it will be interesting to clarify the role of ADAMs and other cell surface proteins in cell-cell fusion events.

Perspectives
ADAM proteins have been shown to be critically involved in two different cell-cell fusion pathways, namely heterotypic sperm-egg fusion and homotypic myoblast-myoblast fusion. This suggests a common mechanism for at least a subset of cell-cell fusion events. Although both fertilin-~ and meltrin-x possess a putative fusogenic peptide within their cysteine-rich domains, it is premature to claim that they are cellular fusion proteins. On the other hand, although they may not mediate fusion per se, they may enact critical prefusion or postfusion steps such as proteolytic remodeling of the extracellular matrix or cell surface, cell-cell adhesion, or the signaling reactions and morphological changes that ensue after two cells fuse. A combination of biochemical and genetic studies is now needed to elucidate the exact roles of fertilin-~, fertilin-13 and meltrin-cx in the pathways of sperm-egg and myoblast-myoblast fusion. In the process, we should advance our general understanding of cell-cell and cell-matrix interactions, and perhaps provide a general mechanism for mammalian cell-cell fusion. Further analyses of fertilin should extend our knowledge about certain forms of infertility and may be useful in designing novel contraceptive agents. Further analyses of meltrin-x should enhance our knowledge of myogenesis, and may advance our understanding of muscle regeneration and certain myopathies.

Acknowledgements
Work in our laboratory was supported by the National Institutes of Health (grant GM48739) to JM White. A-PJ Huovila was supported in part by a grant from the Human Frontier Science Program. We thank B Podbilewicz for sharing unpublished data.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest == of outstanding interest
Rothman JE: Protein machinery of vesicle budding and fusion. Protein Sci 1996, 5:185-194. ;is is the most up-to-date review of protein interactions involved in intracellular vesicle fusion and fission events, The role of SNARE proteins in vesicle targeting, docking, and fusion is discussed in detail. 1.

2. 3. oo

White JM: Membrane fusion. Science 1992, 258:917-924. Wolfsberg TG, Straight PD, Gerena RL, Huovila A-PJ, Primakoff P, Myles DG, White JM: ADAM, a widely distributed and

developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain. Dev Biol 1995, 168:378-383. This paper reports the cloning, sequencing, and characterization both of genes encoding mouse fertilin-(x and -~, and of several sequence-similar cDNAs from guinea pig and mouse testis. The novel gene family was named ADAM. Sequence database searches revealed additional ADAMs from a

698

Cell-to-cell contact and extracellular matrix

variety of mammalian tissues. In situ hybridization and PCR analysis showed that ADAMs are widely expressed in a variety of tissues and that they are developmentally regulated. Wolfsberg TG, Primakoff P, Myles DG, White JM: ADAM, a novel family of membrane proteins containing a disintegrin and metelloprotease domain: multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 1995, 131:275-278. This review describes the ADAMs identified from a variety of organisms. Potential functions of different domains of ADAM proteins, and how these activities may be regulated, are discussed. 5. Zimmerberg J, Vogel SS, Chernomordik LV: Mechanisms of membrane fusion. Annu Rev Biophys Biomol Struct 1993, 22:433-466. Primakoff P, Hyatt H, Tredick-Kline J: Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion. J Cell Bio/1987, 104:141-149. Blobel CP, Wolfsberg TG, Turck CW, Myles DG, Primakoff P, White JM: A potential fusion peptide and an integrin ligand domain in a protein active in sperm-egg fusion. Nature 1992, 356:248-252.
8.

4.

the mouse sperm homolegue of PH-30 (fertUin) beta. J Cell Sci 1995, 108:3267-3278. In this paper, peptide analogs of the fertilin-~ disintegrin loop were demonstrated to inhibit sperm-egg binding and fusion, corroborating other similar results [16",19]. Cloning and sequencing of the gene encoding mouse fertilin-~ are also reported. 19. Fusi FM, Vignali M, Bronson RA: Mammalian oocytes exhibit specific recognition of the RGD (Arg-Gly-Asp) tripeptide and express oolemmal integdns. Mol Reprod Dev 1993, 36:212-219. Bronson RA, Gailit J, Bronson S, Oula L: Echistetin, a disintegrin, inhibits sperm-oolemmal adhesion but not oocyte penetration. Fertil Steril 1995, 64:414-420. Perry ACF, Gichuhi PM, Jones R, Hall L: Cloning and analysis of monkey fertilin reveals novel c( subunit isoforms. Biochem J 1995, 307:843-850. Muga A, Neugebauer W, Hirama T, Surewicz WK: Membrane interaction and conformational properties of the putative fusion peptide of PH-30, protein active in sperm-egg fusion. Biochemistry 1994, 33:4444-4448.

20.

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Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Niewiarowski S: Disintegrins: family of inhibitory proteins from viper venoms. Proc Soc Exp Biol Med 1990, 195:168-171. Hit LA, Jia L-G, Bjarnason JB, Fox JW: cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins. Arch Biochem Biophys 1994, 308:182-191.

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