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A D A M s a n d c e l l f u s i o nA r i - P e k k a J H u o v i l a * t , E d u a r d o A C A l m e i d a * a n d J u d i t h M W h i t e *M e m b e r s o f t h e A D A M f a m i l y ( m e m b r a n e p r o t e i n s w i t h a T a ble 1d i s i n t e g r in a n d m e t a l l o p r o t e a s e d o m a i n ) h a v e b e e n i m p l i c a t e d P o t e n t ia l f u n c t io n s o f A D A M ectodomalns.i n s e v e r a l c e l l - i n t e r a c t i v e e v e n t s , i n c l u d i n g c e l l - c e l l f u s i o n . A D A M S y n o n y m s M e t a l l o p r e t e a s e ? A d h e s i o n ? F u s i o n ?R e c e n t e v i d e n c e im plicat es th ree AD AM s, fertilin-~, fertilin-13,a n d me l t r i n - e q i n sp e r m - e g g fu s i o n a n d my o b l a s t fu s i o n . In A DAM 1 Fertilin -e *A D A M 2 Fert i l in- I~ **l i g h t o f t h e l a r g e n u m b e r a n d w i d e t i s s u e d i s t r i b u t i o n o f t h e A DA M 3 C y ri te s ti n,MDC IA D A M s , t h e y m a y a l s o p a r t i c i p a t e i n o t h e r c e l l - c e l l f u s i o n A DA M 4 *A D A M 5 t M D C II * *e v e n t s . A s A D A M s a r e a l s o f o u n d i n b o t h v e r t e b r a t e s a n d A DA M 6 tM D C IV

A D A M 7 E A P I * *i n v e r te b r a t e s , s o m e f e a t u r e s o f c e l l - c e l l f u s i o n r e a c t i o n s m a y A DA M s M S2b e c o n s e r v e d t h r o u g h o u t t h e a n i m a l k i n g d o m . A DA M 9 M DC 9 **

A D A M 1 0 M P c D N A *A D A M 1 1 M D C * *A D A M 1 2 M e l t r i n - a * *A D A M 1 3A D A M 1 5 * * *

A d d r e s s e s*Depar tment o f Ce l l B io logy , Un ivers ity o f V i rg in ia , Hea l th Sc ien cesCe n te r , P O B o x 4 3 9 , Ch a r lo t te sv il le , V A 2 2 9 0 8 , US AtDe p a r tm e n t o f N e u r o l o g y , Un i ver s it y o f K u o p i o , P O B o x 1 6 2 7 ,RN-70211 Kuop io , F in landSe-mai l : wTg@virginia.eduCo r r e sp o n d e n ce : Ju d ith M W h i teC u r r e n t O p i n i o n i n C e l l B io l o g y 1 9 9 6 , 8 : 6 9 2 - 6 9 9 C u r r e nt B i o l o g y L t d I S S N 0 9 5 5 - 0 6 7 4A b b r e v i a t i o n sA D A M p r o te i n co n ta i n i n g a d i s i n te g r i n a n d me ta l l o p r o te a se d o ma i nS V M P sn a ke ve n o m me ta l l o p r o te a se

I n t r o duc t i o nM e m b r a n e f u s i o n e v e n t s a r e i m p o r t a n t b o t h i n t r ac e ll u l ar l y ,fo r e x a m p le in v e s i c u la r t r a f f i c k in g [1 '] , a n d o n th e c e l ls u r fa c e , fo r e x a m p le in v i ru s -c e l l a n d c e l l - c e l l fu s io n[2 ] . In th i s r e v ie w , w e fo c u s o n c e l l - c e l l fu s io n a n d , i np a r t i c u l a r , o n r e c e n t e v i d e n c e i m p l i c a t i n g m e m b e r s o fa n e w l y d i s c o v e r e d g e n e f a m i l y , t h e A D A M s ( p r o t e i n sc o n t a i n i n g a d i s i n t e g r i n a n d m e t a l l o p r o t e a s e d o m a i n )( [3 " , 4 " ] ; s e e T a b le 1 ) , i n tw o im p o r t a n t c e l l - - c e l l fu s io ne v e n t s , s p e r m - e g g f u s io n a n d m y o b l a s t fu s io n .C e l l - c e l l f u s i o n e v e n t s a r e c r i t i c a l t o t h e d e v e l o p m e n to f m u l t i c e ll u l a r o r g a n i sm s . F o r e x a m p l e , f u s i o n o f s p e r ma n d e g g i n i t i a te s z y g o t i c d e v e l o p m e n t ; f u s i o n o f m y o b l a s t sg e n e ra t e s l a rg e c o n t ra c t i l e u n i t s c a l l e d m y o tu b e s ; fu s io n o fc e r t a i n m o n o c y t e s g e n e r a t e s o s t e o c l a s t s w h i c h a r e b o n e -re s o rb in g c e l ls ; a n d fu s io n o f c y to t ro p h o b la s t s g e n e ra t e s as y n c y t i a l l i n in g o f t h e p l a c e n ta . B e fo re tw o c e l l s c a n fu s e ,h o w e v e r , t h e y m u s t a t t a i n f u s i o n c o m p e t e n c e a n d t h i sin v o lv e s a s e r i e s o f s t e p s a s o u t l in e d in F ig u r e 1 . F i r s t , t h ec e l ls m u s t d i f f e r e n ti a t e , a p r o c e s s t h a t m a y i n v o l v e c h a n g e si n g e n e a n d p r o t e i n e x p r e s s i o n , p r o t e o l y t i c p r o c e s s i n g o fc e l l s u r fa c e p ro te in s , a n d c h a n g e s in c e l l s h a p e (F ig . l a ) .S e c o n d , t h e c e l ls m u s t r e c o g n i z e a n d a d h e r e t o t h e ir f u s i o np a r t n e r ( F i g . l b ) . I n s o m e c a s e s , n o t a b l y i n s p e r m - e g gfu s io n , t h i s i n v o lv e s p r io r c e l l m ig ra t io n . L a s t ly , t h ec e l l s m u s t f u s e , t h a t i s t h e y m u s t m e r g e t h e i r s e p a r a t eb i l a y e r s i n to o n e (F ig . l c ) . A f t e r fu s io n h a s o c c u r re d ,

M e t a r g i d i n , M D C 1 5 ,v a s c u l a r M D C

A n a s t e r i s k i n t h e ' m e t a l l o p r o t e a s e ' c o l u m n i n d i c a te s t h a t t h e s p e c i f ie d A D A M c o n t a i n st h e c o n s e n s u s m e t a i l o p re t e a s e s i t e a m i n o a c i d s e q u e n c e ( t h e H E X X H b o x ) . A l l A D A M Sh a v e b e e n g i v e n a t l e a s t o n e a s t e r i s k i n th e ' a d h e s i o n ' c o l u m n a s t h e y a l l c o n t a i n ap r e d i c t e d d i s i n t e g r in l o o p . T h o s e A D A M s w h o s e p r e d i c t e d d i s i n te g r i n l o o p ' a c t iv e s i te 't r i p e p t i d e c o n t a i n s a n e g a t i v e c h a r g e i n t h e t h i r d p o s i t i o n w h i c h i s c o n s e r v e d i n a llt h e c r e s s - s p e c i e s h o m o l o g s a r e i n d i c a t e d w i t h a d o u b l e a s te r is k . O n e a s t e r i s k i n t h e' f u s i o n ' c o l u m n i n d i c a t e s t h a t t h e s p e c i f i e d A D A M c o n t a i n s a p r e d i c t e d f u s i o n p e p t i d e .R e f e r e n c e s f o r A D A M s 1 - 1 1 a r e g i v e n in [ 3 ' ] a n d t h o s e f o r A D A M s 1 2 a n d 1 5a r e g iv e n i n [ 2 3 " ' ] a n d [ 4 3 ] , r e s p e ct i v e ly . T h e g e n e e n c o d i n g A D A M 1 3 w a s c l o n e df r o m Xenopus/aevis D A l f a n d a ri , T G W o l f s b e r g , J M W h i t e , D D e S i m o n e , u n p u b l i s h e dd a t a ) . E A P , e p i d i d y m a l a p i c a l p r o t e i n ; M D C , m e t a l l o p r o t e i n a s e - l i k e , d i s i n t e g r in - l i k e ,c y s t e i n e - r i c h p r o t e i n .

n e w d e v e l o p m e n t a l p r o g r a m s a r e i n i t i a t e d i n a m a n n e rt h a t p r o b a b l y i n v o l v e s f u s i o n - m e d i a t e d s i g n a l t r an s d u c t i o n(F ig . l d ) .

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1 g96 Cur ren t Op in ion in Ce l l B io logy

Step s in ce l l -c e l l fus ion . In o rder to fuse , ce l ls must : (a ) d i f fe ren t ia teso a s to a t ta in fu s io n co m p e te n ce ; ( b ) a d h e r e to o n e a n o the r ; a n df ina l ly (c) fuse ( i .e . me rge the i r o u te r l ip id b i layers) . A f te r fus ion ,there a re l i ke ly post fus ion s igna l ing events (d ) invo lv ing changes ince l l mo r p h o l o g y a n d p h ys i o l o g y . Th i s f ig u r e d o e s n o t sh o w e i th e rhe te ro typ ic o r hom otyp ic fus ion in par ticu la r ; the fus ing ce l ls a reco l o r e d d i f f e re n t l y o n l y to d i f f e r e n t i a te th e m f r o m e a ch o th e r .

T w o p r i n c i p l e s h a v e g u i d e d o u r w o r k o n c e l l - c e l l f u s i o n .T h e f i r s t i s t h a t , l i k e a l l b io lo g ic a l m e m b ra n e fu s io nr e a c t i o n s , c e l l - c e l l f u s i o n e v e n t s a r e b o t h e n e r g e t i c a l l yu n fa v o ra b le a n d s p e c i f i c [2 , 5 ] ; t h e y w i l l t h e re fo re r e q u i re

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A D A M s en d ce l l f u s io n H u o v i la , A lmeid a an d Wh i t e 693

p r o t e i n s i n o r d e r t o b e c o m e e n e r g e t i c a l l y f a vo r a b le a n dsp ec i f i c . Th e seco n d i s t h a t a s ce l l - - ce l l f u s io n ev en t s a r eto p o lo g i ca l l y eq u iv a l en t t o v iru s -- ce ll f u s io n ev en t s , t h ep ro t e in s t h a t med i a t e ce l l - ce l l f u s io n r eac t i o n s wi l l sh a ret r a it s w i th v i r a l ad h es io n / fu s io n p ro t e in s [2] .A D A M s i n s p e r m - e g g f u s i o nI d e n t i fi c a t i o n o f t h e A D A M sA s e a r c h f o r p r o t e i n s i n v o l v e d in s p e r m - e g g f u s io n t h a tsh a re ru d ime n ta ry t r a i ts w i th v i ra l ad h es io n / fu s io n p ro t e in sled to th e c hara cter iz at ion of fer t i l in [3" ,4" ,5 ] . Fer t i l in i s ah e t e r o d i m e r f o u n d o n t h e s p e r m s u r f a c e a n d i s c o m p o s e do f tw o t y p e I i n te g r a l m e m b r a n e g l y c o p ro t e i ns , n a m e l yfer t i l in - (x and fert il in -13 , the p ro to type s o f the A DA Mfami ly . F e r t i l i n was o r i g in a l l y imp l i ca t ed i n sp e rm-eg gfu s io n o n t h e b as is o f t h e o b se rv a t i o n t h a t an an t i - f e r t i l i nmo n o c lo n a l an t i b o d y i n h ib i t s fu s io n in vitro [6 ] . Thec l o n i n g o f c D N A s e n c o d i n g t h e f e r ti l in s u b u n i ts a sa p p a r e n t l y e x p r e s s e d o n m a t u r e g u i n e a p i g s p e r m r e v e a l e dt h e p r e s e n c e o f a p u t a t i v e d i s in t e g r in d o m a i n i n t h e Is u b u n i t a n d a p u t a t i v e f u s i o n p e p t i d e i n t h e a s u b u n i t[7 ] . Th i s l ed t o t h e h y p o th es i s t h a t f e r t i l i n b in d s t o anin t eg r in o n t h e eg g su r f ace v i a t h e d i s i n t eg r in d o main o fi ts 13 su b u n i t , an d t h a t t h e (x su b u n i t t h en me d ia t es t h eu n i o n o f t h e o p p o s e d p l a s m a m e m b r a n e s .C o m p a r i s o n o f A D A M s w i t h v ir a l f u s io n p r o t ei n sT h e h y p o t h e s i s t h a t f e r t i l i n m a y b e i n v o l v e d i n f u s i o n(F ig . l c sh o ws fu s io n ) i s b ased main ly o n a co mp ar i so no f f e r t il i n wi th v i r a l f u s io n p ro t e in s . F e r t i l i n d i sp l ay s mo s to f t h e c h arac t e r i s t i c s o f vi r al ad h es io n / fu s io n p ro t e in s [2] ,mo s t n o t ab ly a b in d in g d o main i n t h e f e r t i l i n -1 3 su b u n i tan d a p o ss ib l e fu so g en i c m o t i f i n t h e f e r t i l i n - a su b u n i t .Th e b in d in g d o m ain i n t h e f e r t i l in -13 su b u n i t i s i ts' d i s i n t eg r in ' d o main (F ig . 2 ) . D i s in t eg r in s were o r i g in a l l yi so l a t ed f ro m v ip e r v en o ms as sh o r t so lu b l e p o ly p ep t i d est h a t d i s r u p t i n t e g r i n - m e d i a t e d c e l l - c e l l a n d c e l l - m a t r i xin t e r ac t i o n s , h en ce t h e n ame d i s i n t eg r in s [8 ] . S o lu b l e d i s -i n t eg r in s a r e p ro t eo ly t i ca l l y fo rm ed f ro m l a rg e r p r ecu r so r sk n o w n a s s n a k e v e n o m m e t a l l o p r o t e a s e s ( S V M P s ) [ 9 , 1 0 ] .Al l t h r ee su b c l as ses o f S VM P s , n am ely P I , P I I an d P i l l ,c o n t a i n a z i n c - d e p e n d e n t m e t a l l o p r o t e a s e d o m a i n ; P i l lS V M P s a l s o c o n t a i n a f u r t h e r d o w n s t r e a m c y s t e i n e - r i c hd o m a i n . T h e d i s i n te g r i n d o m a i n s o f S V M P s b i n d i n t e g r in swi th h ig h a f f i n i ty an d i n h ib i t i n t eg r in -m ed ia t e d p l a t e l e ta g g r e g a t i o n a n d c e l l - m a t r i x a t t a c h m e n t , t h u s a l l o w i n gs p r e a d o f t h e v e n o m a n d p r o m o t i n g h e m o r r h a g e i n t h eb i te v ict im. As fer t i l in -13 i s a t ransmembrane pro tein ,h o wev er , i t s d i s i n t eg r in d o main i s t h o u g h t t o med ia t e ad i r ec t ce l l - ce l l i n t e r ac t i o n v i a b in d in g t o an i n t eg r in . P I Id i s i n t eg r in s d i sp l ay a 1 3 amin o ac id p ro j ec t i n g l o o p wi ths e q u e n c e s s u c h a s K G D , M V D , a n d R G D ( s i n g l e - l e t t e rco d e fo r amin o ac id s ) a t t h e t i p o f t h e l o o p [1 1 ,1 2 ]. P i l ld i s i n t eg r in s a r e i n s t ead p red i c t ed t o h av e a 1 4 amin o ac idl o o p w i t h s e q u e n c e s s u c h a s E S E C ( s i n g l e -l e t t e r c o d efo r amin o ac id s ) a t t h e i r p r ed i c t ed t i p s . As sh o wn inF ig u re 2 , t h e p r ed i c t e d d i s i n t eg r in l o o p o f mo u se f er ti li n-13

h a s t h e s e q u e n c e Q D E C ( s i n g le - l e t te r c o d e f o r a m i n oac id s ) a t t h e co r r esp o n d in g p o s i t i o n . As d i scu ssed b e lo w,recen t ev id en ce h as imp l i ca t ed t h e p red i c t ed f e r t i l i n -1 3d i s i nt e g r i n l o o p i n s p e r m - e g g b i n d in g .

As f e r ti l in - (x sh a res p ro p er t i e s , m o s t n o t ab ly a p u t a t i v efu s io n p ep t i d e , w i th v i r a l f u s io n p ro t e in s , i t i s co n s id e redto b e a can d id a t e fu so g en i c su b u n i t . V i r a l f u s io n p ep t i d esa r e s h o r t s t r e t c h e s o f m o s t l y h y d r o p h o b i c a m i n o a c i dsfo u n d i n a t r an s me mb ran e su b u n i t o f v i ra l f u s io n p ro t e inc o m p l e x e s . M o s t v i r a l f u s i o n p e p t i d e s s h o w a t e n d e n c yto fo rm a n am p h ip a th i c o t h e l i x an d 13 sh ee t [2] .Th e can d id a t e fu s io n p ep t i d e i n g u in ea p ig f e r t i l i n - (sh a res t h ese ch arac t e r i s t i c s a s we l l a s l imi t ed seq u en cere l a t i o n sh ip wi th an ap o l a r seq u en ce fo u n d i n t h e ru b e l l av i ru s E2 g ly co p ro t e in [7 ] . An o th er p ro min en t f ea tu re o fv i r al f u s io n p ro t e in s i s t h a t t h ey a r e o l i g o mer i c . T h ef e r ti l in c o m p l e x o n t h e s p e r m p l a sm a m e m b r a n e c o n s i s tso f a t i g h t c o m p l e x o f tw o t r a n s m e m b r a n e g l y c o p r o te i nsu b u n i t s [1 3 ] , an d t h ese ap p ear t o b e fu r t h e r o l i g o mer i zed(S I Wate r s , JM W h i t e , u n p u b l i sh ed d a t a ). I n fu r t h e r an a l -o g y wi th v i r a l f u s io n g ly co p ro t e in s , t h e f e r t i l i n su b u n i t sa r e p ro t eo ly t i ca l l y p ro cesse d f ro m l a rg e r p r ecu r so rs [1 3 ].In t e r es t i n g ly , t h e f i n a l p ro t eo ly t i c p ro cess in g ev en t t h a th as b een d e t ec t ed i n f e r t i l i n -1 3 t ak es p l ace co in c id en twi th t h e acq u i s i t i o n o f f e r t i l i za t i o n co mp eten ce ; t h i sp h en o m en o n i s s imi l a r t o t h e ac t i v a t i o n o f th e fu so g en i cp o t en t i a l o f v ir a l f u s io n p ro t e in s b y p ro t eo ly t i c c leav ag e .M o d e l f o r fe r t il i n -m e d i a t e d m e m b r a n e f u s i o nA w o r k i n g m o d e l f o r f e r t i li n - m e d i a t e d m e m b r a n e f u s io nd ep i c t s an i n i t i a l b in d in g i n t e r ac t i o n b e tween f e r t i l i n -1 3a n d a r e c e p t o r o n t h e e g g p l a s m a m e m b r a n e . I t isfu r t h e r p ro p o sed t h a t fo l l o win g b in d in g t h e re i s a co n -fo rmat io n a l ch an g e i n f e r t i l i n t h a t ex p o ses t h e can d id a t efu s io n p ep t i d e i n f e r t i l i n - (x , t h e reb y a l l o win g i t t o b in dh y d ro p h o b i ca l l y t o t h e eg g p l asma me mb ran e [1 4 ] . Byan a lo g y wi th v i r a l mo d e l s [15 ], t h e n ex t s t ep s o f fu s io nare en v i s i o n ed t o i n v o lv e c lu s t e r i n g o f sev era l f e rt i l i nco m p lex es a t t h e fu s io n s i t e , mix in g o f t h e o u t e r l ea f le t so f t h e l i p id b i lay e r s , h emi fu s io n an d , su b se q u en t l y , f u l lfu s io n i n v o lv in g t h e o p en in g an d d i l a t i o n o f a fu s io n p o re[ 15 ]. T h e f u s i o n c o m p l e x o n t h e s p e r m m e m b r a n e c o u l dco n s i s t o f f e r t i l i n su b u n i t s a l o n e , o r i t co u ld co n t a in o th e raccessory pro teins . Al ternat ively , ins tead of fer t i l in - (~ ,a n o t h e r p r o t e i n m i g h t m e d i a t e t h e a c t u a l m e m b r a n efu s io n r eac t i o n .T h e f ir s t p a r t o f t h e h y p o t h e s i s f o r f e r t i li n - m e d i a t e db i n d i n g a n d f u s i o n o f s p e r m a n d e g g i s s u p p o r t e d b y t w ol ines o f ev ide nc e. T h e f i rs t is that an in tegr in ( (x6131) ont h e s u r f a c e o f u n f e r t i li z e d m o u s e e g g s h a s b e e n i d e n t if i e da s a k e y p a r t i c i p a n t i n s p e r m - e g g b i n d i n g [ 1 6 " ' ] . T h esec ond i s that pe p t i de a nalogs o f the fer ti lin -I~ d is in teg r ind o main i n h ib i t sp e rm b in d in g t o eg g s o r t o so mat i c ce l l sthat exp res s t he (x6131 n teg r in [ 16 , 17 ,18"]. In teg r ins havea l so b e en imp l i c a t ed i n t h e f e r t i li za t i o n o f h u m an an dh ams te r eg g s [1 9 ,2 0 ] .

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6 9 4 C e l l - t o - c e l l c o n t a c t a n d e x t r a c e l l u l a r m a t r i x

F i g u r e 2D o m a i n s t r u c t u r e o f A D A M s . F u l l - l e n g t hA D A M p r e c u r s o r s c o n ta i n a s igna ls e q u e n c e ( s s ) , f o l l o w e d b y a prodomain(P ) , a meta l lopro tease - l i ke domain ( M ) ,a d is in tegr in domain (D) , a cys te ine - r ichreg ion ( C ) , a t ransmembrane domain( t m ) , a n d a cy top lasmic ta i l ( t) . The aminoa c i d s e q u e n c e s o f t h e m e t a l l o p r o t e a s eac t ive s i te reg ions and the d is in tegr inl o o p s a r e s h o w n f o r m o u s e f e r t i l i n - ( xand -13 and me l t r in - (x . The can d ida tef u s i o n p e p t i d e r e g i o n s ( in t h e C reg ion)a re sh ow n for mouse fe r t il in -o t andmel t r i n - (~ . T h e a m i n o a c i d s a t t h e ti pof the pre d ic ted d is in tegr in loops a red e n o t e d b y a s t e r i s k s . The puta t ive f u s i o np e p t i d e s a r e i n d i c a t e d b y t h i n h o r i z o n t a ll i n e s . T h e a r r o w ind ica tes the pos i t iono f the pro teo ly tic p rocess ing s tep tha ti s p o s t u l a t e d t o g e n e r a t e t h e ' m a t u r e 'f e r t i l i n -a and f e r t i l i n -13 and 'pu ta t ivem a t u r e ' m e l t r in - c ~ s u b u n i t s . F u s i o n p e p t i d er e g i o n s e q u e n c e s a r e a l i g n e d t o g e t h e ra s p r e s e n t e d in [ 2 3 " ] ; t h e d a s h e s i n t h efer t i l i n s e q u e n c e a r e g a p s s o a s t o a l l o wfo r t h e b e s t a l i g n m e n t o v e r t h e e n t i r e Cd o m a i n . T h e a m i n o t e r m i n u s i s s h o w n a tth e l e f t o f t h e f i g u r e .

s s P M f D

I I I I

Fert i l in-13 CRLAQDECDVTEYCM el tr in -( x C R G S S N S C D L P E F C

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t m t

I I I

1996 Current O p i n i o n n Cell Biology

T h r e e l i n es o f i n d i r e c t e v i d e n c e s u p p o r t t h e h y p o t h e s i sth a t f e r t i l i n -~ s e rv e s a fu s o g e n ic ro l e . F i r s t , c D N A se n c o d i n g f e r t i l i n - ~ s u b u n i t s t h a t h a v e b e e n c l o n e d t od a te , f ro m s e v e ra l d i f f e re n t s p e c ie s [3 , 7, 21 ], c o n ta ina c a n d i d a t e f u s i o n p e p t i d e t h a t c a n b e m o d e l e d a sa n a m p h ip a th i c a h e l ix o r 13 s h e e t . F ig u r e 3 s h o w sh y d r o p h o b i c i t y p l o t s , i n a d d i t i o n t o a - h e l i x a n d ~ - s h e e tp r o j e ct i o n s, o f t h e p u t a t i v e f u s i o n p e p t i d e s o f g u i n e ap i g a n d m o u s e f e r t i l i n - ~ . S e c o n d , t o b e g i n t o t e s t t h ep o s s ib i l i t y th a t t h e p u ta t iv e fu s io n p e p t i d e o f fe rt il in -c ~c a n fu n c t io n a s s u c h , Mu g a e t a l . [2 2 ] s y n th e s i z e d ap e p t i d e c o r r e s p o n d i n g t o t h e p u t a t i v e f u s o g e n i c r e g i o no f g u i n e a p i g f er t il in - c t. T h e p e p t i d e w a s f o u n d t o b i n dt o p h o s p h o l i p i d m e m b r a n e s a n d t o i n d u c e f u s i o n . T h i r d ,a n o t h e r A D A M w i t h a p u t a t i v e f u s i o n p e p t i d e , m e l t r i n - a ,w a s r e c e n t l y im p l i c a t e d in m y o b la s t fu s io n [2 3 ].H o w e v e r , d e s p i t e m u c h s u g g e s t i v e e v i d e n c e , i t i s p r e m a -tu re to c o n c lu d e th a t f e r t i l i n -c t i s a c e l lu l a r fu s io n p ro te in .T h e c r i t i c a l q u e s t i o n i s w h e t h e r t h e c a n d i d a t e f u s i o np e p t id e (F ig s 2 , 3 ) t ru ly fu n c t io n s a s s u c h . A s h a s b e e n th ec a s e fo r v i r a l fu s io n p e p t id e s [24 ], a n s w e r in g th i s q u e s t io nw i ll p r o b a b l y r e q u i r e a c o m b i n a t i o n o f t r a n s f e ct i o n s t u d i e s ,s i t e -s p e c i f ic m u t a g e n e s i s , a n d b i o c h e m i c a l a n d b i o p h y s i c a ls t u d i e s i n c l u d i n g l a b e l i n g w i t h p h o t o a c t i v a t a b l e p h o s p h o -l i p i d l a b e l s . G e n e t i c k n o c k o u t e x p e r i m e n t s m a y a l s o a i dt h e e n d e a v o r . I f f u t u r e w o r k i n d i c a t e s t h a t f e r t i l i n - ~ i s n o ti n v o l v e d i n m e m b r a n e f u s i o n p e r s e ( fu s io n i s s h o w n inF i g . l c ) , t h e n w e w o u l d e x p e c t t h e r e t o b e a n o t h e r p r o t e i no n th e s p e rm (o r e g g ) s u r fa c e th a t fu l f i l l s t h e fu n c t io n o f

m e m b r a n e m e r g e r. T h i s m i g h t b e a n o t h e r A D A M , a s o t h e rA D A Ms a re e x p re s s e d in s p e rm a to g e n ic c e l l s [3 , 2 5 , 2 6 ] ,o r i t m i g h t b e a n o t h e r a n t i g e n i m p l i c a t e d i n s p e r m - e g gfu s io n [2 7 -2 9 ,3 0 ] .I f f e r t i l i n -o c d o e s n o t m e d ia t e fu s io n p e r s e , i t m a y h a v ea n e a r l i e r f u n c t i o n i n t h e p a t h w a y l e a d i n g t o s p e r m - e g gfus io n (F ig . 1 ; [14 ,28 ,30]) . Fo r exa m ple , the p recurso rr e g i o n o f f e r t i l i n - a c o n t a i n s a m e t a l l o p r o t e a s e d o m a i n t h a ti s p re d ic t e d to b e c a t a ly t i c a l ly a c t iv e ( [31 ]; F ig . 2 ) . T h i sm e t a l l o p r o t e a s e d o m a i n c o u l d b e i n v o l v e d i n p r o c e s s i n gc r i t i c a l s p e r m a n t i g e n s d u r i n g s p e r m d i f f e r e n t i a t i o n .A l t e r n a t i v e l y , f e r t i l i n - ~ m i g h t f a c i l i t a t e s p e r m m i g r a t i o nt h r o u g h t h e t e s t i s or r e le a s e o f s p e r m i n t o t h e l u m e n o ft h e s e m i n i f e r o u s t u b u l e s . A s f e r t i l i n - a o n t e s t ic u l a r s p e r mh a s b e e n p r o c e s s e d s o t h a t t h e m e t a l l o p r o t e a s e d o m a i n i sn o l o n g e r c o v a l e n t l y a tt a c h e d , w e d o n o t c u r r e n t l y t h i n kt h a t t h e m e t a l l o p r o t e a s e d o m a i n i s i n v o l v e d i n s p e r mm o v e m e n t t h r o u g h t h e m a l e o r f e m a l e r e p r o d u c t i v e t r a c t s .I t is a ls o p o s s ib l e th a t t h e d i s in t e g r in d o m a in o f f e r t i l i n -~m a y b e i n v o l v e d i n s p e r m - e g g b i nd i n g. A l t h o u g h m a t u r efe r t i l i n -e t a s p u r i f i e d f ro m g u in e a p ig s p e rm d id n o t p o s s e s sa fu l l d i s in t e g r in d o m a in [7 ] , p u r i f i e d m a tu re b o v in ef e r t i l i n - a d o e s ( S I W a t e r s , J M W h i t e , u n p u b l i s h e d d a t a) .G e n e r a l c h a r a c t e r is t i c s o f t h e A D A M sAs me nt ion ed abov e , fe r t il in -oc and fe r t il in -[3 a re th ep r o t o t y p e s o f t h e A D A M g e n e f a m i l y ( s ee T a b l e 1 ).F i g u r e 2 s h o w s t h e g e n e r a l d o m a i n s t r u c t u r e o f A D A M s ,d e d u c e d f r o m th e i r c D N A s e q u e n c e s [ 3 ', 4 ]. N e w l y

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4/8

ADAMs and ce l l fus ion H uov ila , A lme ida and Wh i te 695

F i g u r e 3The putat ive fusion pept ides ofgu inea p ig and m ouse fert il in -(z andmouse melt r in-(x. (a) Kyte- Doo l i t t lehydrophobic i ty p lo ts are shown. Peaksabove the x-axis are hydrophobicregions (e.g. the strongly hydrophobict ransmembrane dom ains). The hor izonta lbars ind icate the posi t ion of thecandida te fusion peptides (fp). tm,t ransmembrane dom ain. The x-axisrepresents posi t ion a long the aminoacid sequence. Th e y-axis representshydrophobic i ty va lue. (b) e . -he l ixp ro jec t ions o f t he AD A Ms a re show n ;shaded areas ind icate possib lehydrophobic faces, and w hi te le t tersind icate hydrophobic amino acids(hydrophobic ity va lues >0.48 on thenormal ized consensus sca le o f E isenberg[44] ) w i th in these areas. The averagehydrophobic it ies o f these faces are 1.3,1.1, and 0.8 for guinea pig fert i l in-cqmou se fert i l in-(x, and m ouse meltrin-cqrespectively. (c) [~-she et projections ofthe ADAMs are shown. Hydrophobicamino acids are h igh l ighted.

(a ) tm i 5'

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Guinea pig fert i l l in-c(

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s y n t h e s i z e d f u l l - l e n g t h A D A M s c o n t a i n a p r o d o m a i n a tt h e i r a m i n o - t e r m i n a l e n d , f o l l o w e d b y a m e t a l l o p r o t e a s e -l i k e d o m a i n . S o m e A D A M s , n o t a b l y f e r t i l i n - o t , h a v e t h ec o n s e n s u s s e q u e n c e f o r t h e a c t i v e s i t e o f a z i n c - d e p e n d e n t

m e t a l l o p r o t e a s e ( H E X X H X X G X X H [ s i n g l e - l e t t e r c o d ef o r a min o a c id s ] ) ; th i s s e q u e n c e i s s imi la r to th o s e f o u n din th e a c t iv e s i t e s o f S VM P s [ 3 ' , 4 ,3 1 ] . A ll AD AM s h a v ea d i s i n te g r i n d o m a i n t h a t m a y b i n d i n t e g r i n s o r o t h e r

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696 Cell-to-cell contact end extracellular matrix

r ecep to r s [3 " ' , 4 " ,3 1 ] . Th e c r i t i ca l amin o ac id r es id u es a tt h e t i p o f t h e p u t a t i v e i n t e g r i n -l i g a n d m o t i f a re i n d i c a t e db y as t e r i sk s i n F ig u re 2 . I n so me ADAM s, n o t ab lyfert il in - (~ , the cy stein e-r ic h reg ion fo l lowing the d is in te -g r in d o m a i n c o n t a i n s a r e l a t i v e ly h y d r o p h o b i c s e q u e n c ethat i s s imi lar to cer tain v i ral fus ion pep t ides (F igs 2 ,3 ) . Al lA D A M c D N A s e n c o d e a p r e d i c t e d m e m b r a n e - s p a n n i n gseq u en c e , fo l l o wed b y a cy to p l asmic t a i l o f v a ry in g l en g th .T h e p r e d i c t e d c y t o p l a s m i c ta il s o f s o m e A D A M s c o n t a i np o ss ib l e r eg u l a to ry s i t e s t h a t may b e i n v o lv ed i n s i g n a l i n g( [3 2 ] ; S I Wate r s , JM Wh i t e , u n p u b l i sh ed d a t a ) .

A D A M s a n d m y o b l a s t f u s i o nT h e f o r m a t i o n o f m y o t u b e s f r o m m y o b l a s ts i s, l i k ef e r ti li z a ti o n , a c o m p l e x m u h i s t e p p r o ce s s . F u r t h e r m o r e ,l i ke s p e r m , p r e f u s i o n m y o b l a s t s m u s t d i f f e r e n t ia t e b e f o r eth ey can fu se . F o l l o win g d i f f e r en t i a t i o n , my o b las t s a l i g nwi th , an d ad h ere t o , each o th e r , an d t h en merg e t h e i rp la s m a m e m b r a n e s t o f o r m m u h i n u c l e a t e d m y o t u b e s[33 ,3 4] . T h e p ro cess o f my o b las t ad h es io n (F ig . l b ) i si t s e l f c o m p o s e d o f a c o m p l e x s e r i e s o f m o l e c u l a r i n t e r -a c t i o n s t h a t h a v e b e e n c l a s s i f i e d a s c a l c i u m - d e p e n d e n to r c a l c i u m - i n d e p e n d e n t [ 3 4 ] . S e v e r a l w e l l c h a r a c t e r i z e dce l l ad h es io n mo lecu l es (CAM s) , i n c lu d in g cad h er in s ,n eu ro n a l an d v ascu l a r en d o th e l i a l CAM s, an d i n t eg r in s ,h a v e b e e n i m p l i c a t e d i n m y o t u b e f o r m a t i o n [ 3 4 ] . H o wmy o b las t s ac tu a l l y fu se an d wh ich ce l l su r f ace p ro t e in s a r ec r i ti c a ll y i n v o l v e d in m e m b r a n e m e r g e r r e m a i n , h o w e v e r ,c h a l l e n g i n g p r o b l e m s . T h e m o s t r e c e n t p r o t e i n im p l i c a t e din my o b las t f u s io n i s an ADAM ca l l ed mel t r i n - (x [2 3 " ' ] .O n t h e b a s is o f t h e o b s e r v a ti o n t h a t t w o m e m b e r s o f t h eAD AM family , fer ti l in -(~ and fert il in -13 , are invo lv ed insp erm- -eg g fu s io n , Yag ami -Hi ro masa e t a l . [2 3 " ' ] s ea rch edfo r f e r t i l i n h o mo lo g s i n mu sc l e ce l l s an d c lo n ed cDNAsen co d in g t h r ee p ro t e in s , meh r in s (x , ~ , an d y . Of t h ese ,t h e e x p r e s s i o n o f m R N A e n c o d i n g m e l tr i n- ( x w a s f o u n d t ob e t h e mo s t r e s t r i c t ed ; i t i s ex p ressed main ly i n sk e l e t a lmu sc l e an d , a t a l o wer l ev e l , in b o n e . (T h e f i n d in g o fm e l t r i n - a i n b o n e i s p o t e n t i a l l y i n t e r e s t i n g b e c a u s e f u s io no f m o n o c y t e s t o f o r m m u h i n u c l e a t e d o s t eo c l a st s o c c u rsi n b o n e . ) A s f u r t h e r e v i d e n c e f o r a r o l e o f m e l t r i n - ai n m y o t u b e f o r m a t i o n , Y a g a m i - H i r o m a s a e t a l . [23" ' ]s h o w e d t h a t w h e n C 2 m o u s e m y o c y t e s a re i n d u c e d t od i f f e r en t i a t e , ex p ress io n o f me l t r i n - (x mRNA in c reases ,wi th a s imi l a r t ime co u r se t o t h a t o f my o g en in , an ea r l ym a r k e r o f m y o b l a s t d i f f e r e n ti a t io n . T h u s , t h e y c o n s i d e r e di t p o ss ib l e t h a t me l t r i n - (x p a r t i c i p a t es i n my o b las t f u s io n .L i k e o t h e r A D A M s , t h e p r e d i c t e d f u l l - l e n g t h m e l t r i n - ( xco n t a in s a me t a l l o p ro t ease d o main an d a d i s i n t eg r ind o ma in . In ad d i t i o n , l i k e f e r t i l i n -~ , me l t r in -o t co n t a in s ap o t en t i a l f u s io n p ep t i d e (F ig s 2 , 3 ) wh ich sh o ws l imi t eds e q u e n c e r e l a t io n s h i p t o th e f u s i o n p e p t i d e o f S e n d a iv i ru s . T h e c a n d i d a t e f u s i o n p e p t i d e o f m e l t r i n - ~ i s,h o w e v e r , l e s s h y d r o p h o b i c t h a n t h e c a n d i d a t e f u s i o npep t ide s o f fer t i l in - (x (F ig . 3 ) .

Y a g a m i - H i r o m a s a e t a l . [23 "] n e x t c o n d u c t ed a fu n c t i o n a lt e s t o f t h e ro l e o f me l t r i n - e t i n my o b las t f u s io n . Wh e n d i f -f e r e n t i a ti n g C 2 m y o c y t e s w e r e t r a n s f e c t e d w i t h f u l l -l e n g thmel t r i n -(x cDN A, fu s io n was s l o wed . Co n v er se ly , wh e na s h o r t e n e d v e r s i o n o f m e l t r i n - a l a c k i n g t h e p r o d o m a i nan d meta l l o p ro t ease d o main (F ig . 2 ) was t r an s fec t ed i n toth e same ce l l s , f u s io n was acce l e r a t ed an d t h e fu s io np ro d u c t s were 'my o sacs ' , r o u n d s t ru c tu res wi th i r r eg u l a r l yd i s t r i b u t ed n u c l e i , i n s t ead o f t h e ch arac t e r i s t ic e l o n g a t edm y o t u b e s w i t h a l i g n e d n u c l e i . M y o s a c s h a v e p r e v i o u s l yb e e n s h o w n t o f o r m a f t e r t h e a p p l i c at i o n o f m i c r o t u b u le -d i s t u r b i n g a g e n t s a n d P M A ( p h o r b o l m y r i s t a t e a c e t a t e ) ,a p ro t e in k in ase C ac t i v a to r , t o d ev e lo p in g my o b las t s .M e l t r i n -~ h as a r e l a t i v e ly l arg e cy to p l asmic t a i l t h a t co u ldb e i n v o lv ed i n i n t e r ac t i o n s wi th in t h e fu s in g ce l l s , su chas wi th cy to sk e l e t a l o r s i g n a l i n g mo lecu l es . Hen ce i t wassu g g es t ed t h a t o n e fu n c t i o n o f me l t r i n - e t mig h t b e t o h e lpr e g u l a te m y o t u b e m o r p h o l o g y [ 35 ].

T h e o p p o s i t e e f f e c t s o f fu l l - l en g th mel t r in - (~ an d t h e co n -s t r u c t la c k i n g th e p r o d o m a i n a n d m e t a l l o p r o t e a s e d o m a i ns u g g e s t e d t h a t p r o t e o l y t i c p ro c e s s i n g m a y b e i m p o r t a n t i nr eg u l a t i n g t h e fu n c t i o n ( s ) o f me l t r i n - (x . Yag ami -Hi ro masae t a l . [2 3 " ' ] sh o wed t h a t d ev e lo p in g mu sc l e ce l l s co n t a inb o th t h e ' f u l l - l en g th ' v e r s i o n o f me l t r in - (x an d sm al l e rfo rms . T h e smal l e r fo rms co u ld co r r esp o n d t o v e r s io n s t h a tl ack , p ro g ress iv e ly , t h e p ro d o m ain an d t h e m eta l l o p ro t eased o main . Yag ami -Hi ro masa e t a l . d id n o t , h o wev er , ev a lu a t ew h i c h f o r m s o f m e l t r i n - ~ a r e e x p r e s s e d o n t h e s u r fa c eo f d ev e lo p in g my o b las t s . By an a lo g y t o f e r t i l i n , t h es m a l l e st f o r m o f m e l t r i n - ~ ( 5 8 k D a ) c o u l d b e t h e ' m a t u r ef o r m ' c o n t a i n i n g t h e d i s i n t e g r i n a n d d o w n s t r e a m r e g i o n s(Fig . 2 , r igh t o f the ar row) and cou ld , po ten t ial ly , bein v o lv ed i n my o b las t ad h es io n , fu s io n , o r s i g n a l i n g . Th el a rg e s t f o r m c o n t a i n i n g t h e u p s t r e a m p r o d o m a i n a n dm e t a l l o p r o t e a s e d o m a i n c o u l d b e a p r e c u r s o r i n w h i c hth e d o wn s t r eam fu n c t i o n s o f me l t r i n - (~ ( e .g . p ro t eo ly s i s ,ad h es io n , fu s io n , o r s i g n a l i n g ) a r e mask ed . Ex p ress io n o ft h e p r e c u r s o r m i g h t p r e v e n t m y o b l a s t s f r o m a n o m a l o u sp r e m a t u r e f u s i o n . A n i n t e r m e d i a t e f o r m i n w h i c h t h ep r o d o m a i n , b u t n o t t h e m e t a l l o p r o t e a s e d o m a i n , h a s b e e nr e m o v e d c o u l d b e i n v o l v e d i n p r o t e o l y t i c e v e n t s t h a ta r e p re r eq u i s i t e s t o my o b las t f u s io n . Yag ami -Hi ro masae t a l . [2 3 " ' ] sp ecu l a t e t h a t me l t r i n -(x co u ld b e i n v o lv edin d eg rad in g t h e ex t r ace l l u l a r ma t r i x . M el t r i n - a ma ya l so b e i n v o lv ed i n p ro t eo ly t i ca l l y ac t iv a t i n g i t s e l f o ro th e r my o b las t su r f ace p ro t e in s . I n b o th v i r a l f u s io na n d s p e r m - e g g f u s io n , p r o t e o l y t i c p ro c e s s i n g o f s u rf a c ean t i g en s i s e s sen t i a l f o r su b seq u en t fu s io n [2 ] .

T ran s fec t i o n o f me l t r i n -~ i n to f i b ro b las t s d id n o t p ro mo tead h es io n o r fu s io n , su g g es t i n g t h a t me l t r i n - e ( a l o n e d o esn o t f u n c t i o n a s a h o m o t y p i c a d h e s i o n o r f u s i o n m o l e c u l e[2 3 " ' ] . As t h e au th o r s su g g es t ed , me l t r i n - (~ may ex i s ta s p a r t o f a h e t e r o d im er (o r h ig h er o l i g o mer ) a s d ofer t i l in -o t and fer t i l in -13 , o r i t may need o ther factorst o e x e r t i t s f u n c t io n . T h e p r e s e n c e o f a d i s in t e g r in - l ik e

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A D A M s a n d c e l l f u s i o n H u o v i la , A l m e i d a a n d W h i t e 6 9 7

d o m a i n s u g g e s t s t h a t m e l t r i n - a m a y b e i n v o l v e d inc e l l a d h e s i o n , w h i c h i s c o n c e i v a b l e a s m y o b l a s t s e x p r e s si n t e g r i n s o n t h e i r p l a s m a m e m b r a n e [ 3 6 , 3 7 ] . T h e s h o r tf o r m o f m e l t r i n - a [ 2 3 ] c o n t a i n s t h e e n t i r e d i s i n t e g r in - l i k ed o m a i n , b u t t h e s e q u e n c e a t t h e a p e x o f t h e p u t a t i v ed i s i n t e g r i n l o o p ( a m i n o a c i d s e q u e n c e S N S C ; s e e F i g . 2 )d o e s n o t c o n ta in a n a c id ic r e s id u e a t t h e th i rd p o s i t io n( as d oe s , fo r e x a m p l e , t h e s e q u e n c e Q D E C i n m o u s efe r t i l in -~ ) , a c o m m o n fe a tu re o f fe rt il in -1 3 a n d P i l ls n a k e v e n o m h e m o r r h a g i c d i si n te g ri n s. T h e s e q u e n c er e q u i r e m e n t s f o r a f u n ct i o n a l d i s i n t e g r in l o o p r e m a i n ,h o w e v e r , t o b e d e t e r m i n e d f o r t h e c a s e s o f b o t h i n t e g r ina n d p o t e n t i a l n o n i n t e g r i n [ 38 ] r e c e p t o r s. T h u s , m e l t r i n - ~c o u ld b in d to a n in t e g r in o r a n o n in te g r in r e c e p to r o n i tsfu s io n p a r tn e r .I n s u m m a r y , it h a s b e e n d e m o n s t r a t e d t h a t m e l t r i n - ~ i si n v o l v e d i n t h e p a t h w a y l e a d i n g t o m y o b l a s t f u s io n [ 23 ].As i s th e c a s e fo r f e r t i l i n , h o we v e r , t h e re i s a s y e t n oe v i d e n c e i m p l i c a t i n g m e l t r i n - c ~ i n m e m b r a n e f u s i o n p e rse. T h e p o s s ib le ro le s o f me l t r in -c t i n p ro te o ly s i s , c el la d h e s io n , c e l l fu s io n , a n d c e l l s ig n a l in g mu s t n o w b et e s t e d . W h e t h e r o r n o t m e l t r i n - ~ i s i n v o l v e d i n f u s i o nper se , i t i s l i k e ly th a t a d d i t io n a l p ro te in s , p e rh a p s o th e rA D A M s [ 3 " ] , a r e n e e d e d t o o r c h e s t r a t e t h e c o m p l e xm o l e c u l a r i n te r a c t i o n s t h a t l e a d to m y o b l a s t - m y o b l a s tfu s io n .

PerspectivesA D A M p r o t e i n s h a v e b e e n s h o w n t o b e c ri t ic a l ly i n v o l v e di n t w o d i f f e r e n t c e l l - c e l l f u s i o n p a t h w a y s , n a m e l y h e t -e ro ty p ic s p e rm-e g g fu s io n a n d h o mo ty p ic my o b la s t -my o b la s tf u s io n . T h i s s u g g e s t s a c o m m o n m e c h a n i s m f o r a t le a s t as u b s e t o f c e l l - c e l l f u s i o n e v e n t s . A l t h o u g h b o t h f e r t i l i n - ~a n d m e l tr in - x p o s s e s s a p u ta t iv e fu s o g e n ic p e p t id e w i th int h e i r c y s t e i n e - r i c h d o m a i n s , i t i s p r e m a t u r e t o c l a i mth a t th e y a re c e l lu l a r fu s io n p ro te in s . On th e o th e rh a n d , a l t h o u g h t h e y m a y n o t m e d i a t e f u s i o n per se, t h e ym a y e n a c t c r i t i c a l p r e f u s i o n o r p o s t f u s i o n s t e p s s u c h a sp r o t e o l y t i c r e m o d e l i n g o f t h e e x t r a c e l l u l a r m a t r i x o r c el ls u r fa c e , c e l l - c e l l a d h e s io n , o r th e s ig n a l in g re a c t io n s a n dm o r p h o l o g i c a l c h a n g e s t h a t e n s u e a f t e r t w o c e l l s f u s e .A c o m b i n a t i o n o f b i o c h e m i c a l a n d g e n e t i c s t u d i e s i sn o w n e e d e d t o e l u c i d a t e t h e e x a c t r o l e s o f f e r ti l i n -~ ,fer ti li n-13 a n d me l t r in -c x in th e p a th w a y s o f s p e r m -e g ga n d m y o b l a s t - m y o b l a s t f u s io n . I n t h e p r o c e ss , w e s h o u l da d v a n c e o u r g e n e r a l u n d e r s t a n d i n g o f c e l l - c e l l a n dc e l l - m a t r i x i n t e r a c t i o n s , a n d p e r h a p s p r o v i d e a g e n e r a lm e c h a n i s m f o r m a m m a l i a n c e l l- c e l l f u si o n . F u r t h e r a n a l y -s e s o f fe r t il i n s h o u ld e x t e n d o u r k n o w l e d g e a b o u t c e r t a i nf o r m s o f in f e r t i li t y a n d m a y b e u s e f u l i n d e s i g n i n g n o v e lc o n t r a c e p t iv e a g e n t s . F u r th e r a n a ly s e s o f me l t r in - x s h o u lde n h a n c e o u r k n o w l e d g e o f m y o g e n e s is , a n d m a y a d v a n ceo u r u n d e r s t a n d i n g o f m u s c l e r e g e n e r a t i o n a n d c e r t a inm y o p a t h i e s .

G i v e n t h e l a r ge n u m b e r o f A D A M s a n d t h e i r w i d ed i s t r ib u t io n [3 , 4 ], i t i s l i k e ly th a t AD AM s p a r t i c ip a tein o th e r c e l l - - c e l l fu s io n e v e n t s . In a d d i t io n to f e r t i l i n -aa n d m e l t r in -c x (AD AM s 1 a n d 1 2) , AD AM s 9 a n d 1 1c o n t a i n p u t a t i v e f u s i o n p e p t i d e s [ 3 , 4 ] . O n e A D A M ,A D A M 8 , w a s c l o n e d f r o m m a c r o p h a g e s w h i c h f o r mm u l t i n u c l e a t e d g i a n t c el l s d u r in g i n f l a m m a t o r y r e a ct i o n s[ 3 9 ] . A D A M c D N A s a r e a l s o e x p r e s s e d i n t h e p l a c e n t a ,w h e r e c y t o t r o p h o b l a s t s f u s e t o f o r m s y n c y t i o t r o p h o b l a s t s( G e n B a n k a c c e s s n u m b e r H 1 2 6 2 7 ; P D S t r a i g h t , J M W h i t e ,u n p u b l i s h e d d a ta ) . Me l t r in -c t i s a l s o e x p r e s s e d in b o n e ,w h e r e m o n o c y t e s f u s e t o f o r m o s t e o c l a s t s .A D A M c D N A s h a v e b e e n c l o n e d f r o m Xenopus ( A D A M 1 3 ;D A l f an d a r i, T G W o l f s b er g , J M W h i t e , D D e S i m o n e ,u n p u b l i s h e d d a t a ) , Drosophila ( G e n B a n k a c c e s s n u m b e rG0 1 2 0 4 ) , a n d Caenorhabditis elegans ( A D A M 1 4; B P o d -b i l e wic z , p e r s o n a l c o mmu n ic a t io n ; s e e a l s o [4 ] ) . T h e f i r s tA D A M f o u n d i n C. elegans (ADAM 1 4 ) i s p re s e n t in c e i l sth a t u n d e rg o fu s io n re a c t io n s in th e h y p o d e rmis ( [4 0 ] ;B P o d b i le w i c z , p e r s o n a l c o m m u n i c a t i o n ) . A s A D A M s a r ef o u n d i n l o w e r a n i m a l s i n c e l l s t h a t u n d e r g o c e l l - c e l lfu s io n , i t a p p e a r s p l a u s ib le th a t a t l e a s t s o me fe a tu re so f c e ll - c e l l f u s i o n r e a c t i o n s a r e c o n s e r v e d t h r o u g h o u tt h e a n i m a l k i n g d o m . T h e r e m a y , h o w e v e r , b e i m p o r t a n te x c e p t i o n s . C e l l s u r f a c e p r o t e i n s t h a t a r e u n r e l a t e dt o A D A M s h a v e b e e n i m p l i c a t e d i n s e a u r c h i n a n dChlamydomonas f e r t i l i z a t io n [4 1, 42 ]. AD AM s a re a l so n o tl i k e l y t o b e i n v o l v e d i n y e a s t m a t i n g , b e c a u s e t h e g e n o m eo f Saccharomyces cerevisiae d o e s n o t a p p e a r t o c o n t a i nA D A M g e n e s ( T G W o l f s b e r g , p e r s o n a l c o m m u n i c a t i o n ) .T h u s , i t w i l l b e i n t e r e s t i n g t o c l a r i fy t h e r o l e o f A D A M sa n d o th e r c e l l s u r fa c e p ro te in s in c e l l - c e l l fu s io n e v e n t s .A ckn o w l ed g em en tsWork in our laboratory was supported by the National Insti tutes of Health(grant GM48739) to JM White. A-PJ Huovila was supported in part by agrant from the Huma n Fron tier Science Program. We thank B Podbilewiczfor sharing unpublished data.

References and recommended readingPapers of par t icu lar in teres t , publ ish ed wi th in the annua l per iod of rev iew,hav e been h i gh l i gh t ed as :

o f s pec ia l i n t e r es t= = o f ou t s t an d ing in t e r es t1 . R o t h m a n J E : P r o t e i n m a c h i n e r y o f v e s i c l e b u d d i n g a n d f u s i o n .

Protein Sci 1 9 9 6 , 5 : 1 8 5 - 1 9 4 .; i s i s the most up - t o - da t e r ev i ew of protein interact ions invo lved in int racel-lu lar ves ic le fus ion and f ission events, The role of S NA RE p r o t e i ns in vesiclet a r ge t ing , doc k ing , an d f us i on is discussed in detai l .2 . W h i t e J M : M e m b r a n e f u s i o n . Science 1 9 9 2 , 2 5 8 : 9 1 7 - 9 2 4 .3. Wo l f sberg TG, St ra igh t PD, Ge rena RL, Huo v i la A-PJ , Pr imakof f P ,oo M y les DG , W h i t e J M : A DA M , a w id e l y d i s t r i b u t e d a n dd e v e l o p m e n t a l l y r e g u la t e d g e n e f a m i l y en c o d i n g m e m b r a n ep r o t e i n s w i th a d i s i n te g r i n a n d m e t a l l o p r o t e a s e d o m a i n . D ev

Bio l 1 9 9 5 , 1 6 8 : 3 7 8 - 3 8 3 .This pape r r epo r t s t he c l on ing , s equenc ing , and characterization both ofgenes encoding mouse fert i l in-(x and -~, and of several sequence-similarc DNA s f r om gu inea p i g and m ouse tes t is . The nov e l gene fami ly was nam edA D A M . S e q u e n c e database searches revea led add i tiona l AD AM s from a

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variety of mam malian issues. In situ hybridization and PCR analysis showedthat ADAMs are widely expressed in a variety of t issues and that they aredevelopmental ly regulated.4. Wolfsberg TG, Primakoff P, Myles DG, White JM: A D A M , a nove l fami l y o f mem brane p ro te i ns con ta i n i ng a d i s i n teg r in andme te l l op ro tease doma in : m u l t i po ten t i a l func t i ons i n ce l l - ce l l

and ce l l -ma t r i x i n te rac t ions . J Ce l l B i o l 1995, 131:275-278.This review describes the AD AMs identi fied from a variety of organisms.Potential functions of di fferent domains of ADAM proteins, and how theseactivit ies ma y be regulated, are discussed.5. Zimmerberg J, Vogel SS, Chernomordik LV: Mechanisms ofmem brane fus i on . Annu R ev B iophys B iomo l Struct 1993,22 :433 -466 .6. Primakoff P, Hyatt H, Tredick-Kl ine J: Identi f icat ion andpur i f ica t ion o f a sperm sur face pro te in wi th a potent ia l ro le ins p e r m - e g g m e m b r a n e f u s io n . J C e l l B i o /1987 , 104 :141 -149 .

Blobel CP, Wolfsberg TG, Turck CW, M yles DG, Primakoff P,Whi te JM: A potent ia l fus ion pept ide and an in tegr in l iganddoma in i n a p ro te i n ac t i ve in sp e rm -egg fus i on. Nature 1992,356 :248 -252 .8 . Gould R , Polokoff MA, Friedman PA, Huang TF, Holt JC, Co ok J ,Niewiarowski S: Dis in tegr ins: fami ly o f inh ib i tory pro te insf rom v i pe r venoms. Proc Soc Exp B io l Med 1990, 195:168-171.

Hi t LA, Jia L-G, B jarnason JB, Fox JW: cD NA sequ ences fo r fou rsnake v enom me ta l l op ro te i nases : s t r uc tu re , c l ass i fi ca t ion , andthe i r r e l a t i onsh ip to mam ma l i an rep roduc t i ve p ro te i ns . ArchBiochem Biophys 1994, 30 8 :182 -191 .10 . B ja rnason B , Fox J : Snake venom me ta l l oendopep t i dases :reprop lys ins. Methods Enzymol 1995, 248:345-368.11 . Adler M, Lazarus RA, Dennis MS, Wagner G: S o lu t ion structureof k is trin , a po tent p la te le t aggreg at ion inh ib i tor and G P l i b - I l i aantagonist . Science 1991, 253 :445 -448 .12 . Saudek V, Atk inson R A, Pe l ton JT: Three-d ime nsiona l structureof ech ista t in , the smal lest act ive RGD prote in . Biochemistry

1991, 30:7369-7372.13 . Blobel CP, Myles DG, Primakoff P, White JM: Proteolyticp rocess ing o f a p ro te i n invo l ved in spe rm-eg g fus i oncorre la tes wi th acqu is i t ion o f fer t i l i za t ion competence. J Ce l lB i o l 1990, 111:69-78.14. Snel l WS, White JM: The mo lecu les o f mam mal ian fer t i l i za t ion.Ce l l 1996, 85:629-637.his is the most up-to-date review of molecular interactions in mammaliansperm-egg adhesion and fusion. The interactions between sperm and theegg zona pel lucida, in addition to the interactions between sperm and eggplasma mem branes, are discussed in detai l .15. Whi te JM: Mem brane fusion: the in f luenza parad igm. C o l dSpring Harbor Syrup Quant Biol 1995, 60:581-588.16. Aim ida EA C, Huovi la A-PJ, Suthertand AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P,Myles DG, Whi te JM: Mouse egg in tegr in C (6~ func t i ons as spe rm recep to r. Ce l l 1995, 81 :1095 -1104 .This paper reports the identi fication o f m ouse egg integrin (x6~1 as a spermreceptor. Sperm bound to c(6J31 on unferti lized eggs, and also to somaticcel ls expressing c(6~1. Inhibition of this binding by peptide analogs of thefertilin-13 disintegrin domain implicated fertilin in o6~l-mediated sperm-eggbinding. This is the first report of ce l l-cel l interaction m ediated by integrinc(6~1, for which the only know n l igands unti l now h ave been extracel lular ma-tr ix laminins. The paper proposes that binding between ferti l ins and integrinsmay provide a general m echanism for m amm alian gamete adhesion.17. Myles DG, Kimmel LH, Blobel CP, White JM, Primakoff P:Ident i f i ca t ion o f a b ind ing s i te in the d is in tegr in domain o ffe r t il i n r equ i red fo r s pe rm -egg fus ion . Proc Na t /Acad Sc i USA1994, 91:4195-4198.18 . Eva ns P , Schul tz RM, Kop f GS : Mouse spe rm -egg p l asma mem brane i n te rac t ions : ana l ys i s o f r o l es o f eg g i n teg r i ns and

the m ouse sp e rm h omo legue o f PH-30 ( fer tU in) beta . J Ce l lS c i 1995, 108 :3267 -3278 .In this paper, peptid e analogs of the fertil in-~ disintegrin loop were demon-strated to inhibit sperm-egg binding and fusion, corroborating other similarresults [16",19]. Cloning and sequencing of the gene encoding mousefertilin-~ are also reported.

19. Fusi FM, V igna li M, Bronson RA: Mam mal ian oocytes exh ib i tspec i f i c recogn i t i on o f the RGD (A rg -G l y -Asp ) t r i pep t i deand exp ress oo lemma l i n tegdns . Mol Reprod Dev 1993,36 :212 -219 .20. Bronson RA, Gail i t J, Bronson S, Oula L: Echistetin, a disinte grin,i nh ib i t s spe rm-oo lemma l adhes ion bu t no t oocy te pene t ra t i on .Fertil Steril 1995, 64 :414 -420 .21. Perry ACF, Gichuhi PM, Jones R, Hal l L: Clo ning and analysis ofmonk ey fe r t i li n r evea l s nove l c ( subun i t i so fo rms . Biochem J1 9 9 5 , 307 :843 -850 .22. Muga A, Neugebauer W, Hi rama T, Surewicz WK : Mem branein te rac ti on and con fo rma t i ona l p rop e r t i es o f the pu ta t i vefus i on pe p t i de o f PH-30 , p ro te i n ac t ive i n spe rm -egg fus i on .Biochemistry 1994, 33 :444 4 -4448 .23. Yagami-Hiromasa T, Sato T, Kurisaki T, Kamijo K, Nabeshima Y, . Fu j i sawaseha ra : A me te l l op ro tease -d i s i n teg r i n pa r t i c ipa t i ng i nm y o b l a s t fus ion. Nature 1995 , 377 :652 -65 6 .This pape r reports identification of three fertilin homo logs, meltrins ~ ~, andy, in myo genic cel ls, and further characterizes meltr in-(x as exp ressed in C2myocytes. Meltr in-(x was shown to be cri t ically involved in myo tube formation,but w hether i t is directly involved in plasma mem brane fusion per se, or hasanother function in myotube formation, remains to be investigated. As thisferti l in-related protein is involved in myogenesis, the authors suggest thatthere may exist common mechanisms for spe rm-eg g and myoblast fus ion.24 . Durrer P, Gaudin Y, Ruigrog RW H, Graf R , Brunner J:Pho to l abe l i ng i den ti f i e s a pu ta t i ve fus i on doma in i n theenve lope g l ycop ro te i n o f r ab ies and ves i cu la r s toma t i t i sv i ruses. J B i o / C h e m 1995, 270 :17575 -175 81 .25 . L inder B, Bam mer S, Hein le in UAO: Delaye d transla t ion

and pos t t r ans la t iona l p rocess ing o f cy r i tes t in , an i n teg ra lt r ansmembrane p ro te i n o f the mouse ac rosome . Exp Cel l Res1995, 221:66-72.26 . Carrol DJ, Dikegoros E, Koppel DE, Cowan AE: Surfaceexp ress ion o f the p re -be te subun i t o f fe r t i l in i s r egu la ted a ta post- t ransla t iona l leve l in gu inea p ig spermat ids. Dev B io /1995, 168:429-437.27. My les DG: Mo lecu la r mecha n i sms o f spe rm-e gg mem braneb ind ing and fus i on i n mam ma ls . Dev B io l 1993, 158:35-45.28 . Yanagimachi R: Mam mal ian fer t i l i za t ion. In Physiology ofReproduction. Edited by K nobi l E, Nei l l JD. New York: RavenPress; 1994:189-317.29 . A l len CA, Green DP: Monoclona l ant ibod ies recognize equ i tor ia ls e g m e n t e p i t o p e s p r e s e n t ed de novo fo l l ow ing th e A23187 -i nduced ac rosom e reac t i on o f the gu inea p i g spe rm. J Ce l l Sc i1995, 108:767-777.30 . Wassa rman P : Towards a mo lecu la r mech an i sm fo r game te , adhes ion and fus i on du r i ng mam ma l i an fe r t il i za ti on . Curr OpinCel l B io11995, 7 :658 -664 .This review discusses sp erm -eg g interactions during ferti lization, both at thezona pel lucida and at the plasma mem brane. It is propo sed that in mam-malian ferti lization, adhesion at the zona pel lucida is based on carbohydrateinteractions, and that plasma membrane adhesion and fusion are integrin-mediated.31 . Wolfsberg TG, B azan FJ, Blobel CP, Myles D G, P rimakoff P,Whi te JM: The precursor reg ion o f a pro te in act ive ins p e r m - e g g f u s io n c o n t a in s a m e t a l l o p ro t e a s e d o m a i n :structura l , funct iona l , and evo lu t ionary impl ica t ions. Proc Nat/Acad Sc i USA 1993, 90:10783-10?87.

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