The nuc leo t ide sequence of the human ca t ion i n d e p e n d e n t M6P recep to r (CI-MPR)
and the IGF II r e cep to r are 99.8 % iden t i ca l (1, 2). This sugges t s t h a t one
po lypep t ide s e r v e s as a r ecep to r for M6P-con ta in ing g lycopro te ins such as
lysosomal enzymes and for the IGF II. It has been shown t h a t an t ibod ies a g a i n s t
the CI-MPR cross r eac t with the IGF II r ecep to r (1, 3), and t h a t the CI-MPR and
f ragments of the CI-MPR p re sen t in serum and ur ine bind IGF II (4). The two
l igands do not compete for b inding (3, 5) and a n t i s e r a ra i sed aga in s t the CI-
MPR inh ib i t b inding of the two l igands in a d i f f e r e n t i a l manner (5). These
o b s e r v a t i o n s sugges t t h a t M6P and IGF tI bind a t d i f f e r e n t s i t e s of the recep tor .
In the p r e sen t s tudy we repor t on the binding of a M6P neoglycoprotein , and
of IGF II to d i f f e r e n t t ryp t i c f r agments of the recep tor . In addi ton we p rov ide
ev idence t h a t a s ingle r ecep to r can bind s i m u l t a n e o u s l y M6P and IGF II.
Materials and Methods
Homogeneous human l i v e r M6P/IGF II r ecep to r and i t s a n t i s e r a were ob ta ined as descr ibed (6). PMP-BSA was p repared as descr ibed (7) and i t s an t i s e rum was ra i sed in rabbi t . IgG were i so l a t ed using p ro te in A - S e p h a r o s e 4B (Pharmacia)
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column. Phosphomannan from Hansenu la ho l s t i i was a g i f t from Dr. M. Slodki (U.S.D.A. Nor thern Regional Research Center , Peoria, I1.). Apro t in in was from Boehringer , Mannheim, t r i l y s i n e pep t ide from Serva , Biogel P - 4 and P - 3 0 from Biorad, d i succ in imidy l s u b e r a t e (DSS) from Pierce Chem, Co., and TPCK t r e a t e d t ryps in were ob ta ined from Sigma Chem. Co. IGF II ( p r epa ra t i on II/4) and a mix tu re of IGF I a n d IGF II ( p r epa ra t i on 1932) were a g i f t from R.E. Humbel, Ztirich. IGF II was iod ina ted with Na~25I us ing the ch loramine T procedure (8). The spec i f ic a c t i v i t y of [~2~I]IGF II was 112,000 cpm/ng. Goat an t i r abb i t IgG- a lka l ine p h o s p h a t a s e conjuga te was from TAGO. Other r e a g e n t s used in th i s s tudy were of a n a l y t i c a l grade.
Apro t in in s u b s t i t u t e d with p e n t a m a n n o s e 6 - p h o s p h a t e and t r i l y s i n e [PMP- l y s - a p r o t i n i n ] : Pen tamannose 6 - p h o s p h a t e (PMP) as a sodium sa l t was ob ta ined from phosphomannan as descr ibed (9). Apro t in in (15 mg/ml) was i ncuba t ed with PMP (200 mg/ml) in 50 mM N, N - bis ( 2 - h y d r o x y e t h y l ) g lycine , pH 9.0 and 160 mM NaCNBHa at 37°C for 24 h (10). The PMP-apro t in in was pur i f ied us ing a Biogel P - 4 column (1.5 x 28) in 50 mM ace t ic acid and c a r b o h y d r a t e con t en t was de te rmined (11). The PMP-apro t in in was coupled th rough carboxyl ic groups with t r i l y s i n e pep t ide (13). P M P - l y s - a p r o t i n i n was iod ina ted with Na [~2~I] us ing the ch loramine T procedure (8) and pur i f ied th rough Biogel P - 4 and P - 3 0 columns. The spec i f ic a c t i v i t y was of 84,000 cpm/ng pro te in .
T ryp t i c d iges t ion of the recep to r : M6P/IGF Ii r e cep to r p r epa ra t i on (0.4 mg/ml) was i n c u b a t e d a t 37°C with TPCK t r e a t e d t r yps in a t r ecep to r to t ryps in ra t io of 60:1 (w/w) in 50 mM sodium phospha t e pH 7.5 con ta in ing 150 mM NaC1 and 0 . 1 % Tr i ton X 100 (buffer A) for 12-24 h. Tryp t ic pep t ides were c h a r a c t e r i z e d by SDS-po lyac ry lamide gel e l e c t r o p h o r e s i s (14) fol lowed by s i l v e r s t a i n i n g or Western b lo t t i ng (15).
Binding and cross l ink ing of P M P - l y s - a p r o t i n i n and IGF II wi th r e c e p t o r and i t s t r y p t i c pep t ides : The ace tone p r e c i p i t a t e of 1 ~lg pur i f ied r ecep to r or of t r yp t i c pep t ides de r ived from 2.1 ug recep to r were so lub i l i zed in 14 ~1 buf fe r A and i n c u b a t e d for 30 min a t 4°C with and wi thou t 5 mM mannose 6 - p h o s p h a t e or g lucose 6 - p h o s p h a t e or 10-6M IGF II. [12~I]PMP-lys-aprotinin, 800,000 cpm or [125I]IGF II, 400,000 cpm in 16 111 buf fe r A were added and incuba t ed for 3 h at 4°C. Cross l ink ing of bound P M P - l y s - a p r o t i n i n or IGF II with r ecep to r was car r ied out by i ncuba t ing the r eac t i on mix ture for 15 min a t 4°C with 0.06 mM DSS. When binding of P M P - l y s - a p r o t i n i n was performed in the p resence or absence of 5 mM lys ine , cross l ink ing was done with 0.6 mM DSS. Unreac ted DSS was quenched with 20 111 of 2 5 0 mM Tris-HC1 pH 7.5. The samples were immunoprec ip i t a t ed (16) and sub jec ted to SDS-po lyacry lamide gel e l e c t ropho re s i s fol lowed by au to rad iog raphy .
Immunoprec ip ia t ion of M6P/IGF II r e c e p t o r - l i g a n d complexes wi th a n t i PMP- BSA an t ibod ies : All r e a g e n t s were in buf fe r A. Pur i f ied receptor , 0.2 ng in 47 til, was i n c u b a t e d with and wi thou t IGF II, 4 lag in 4 111 for 30 min a t 4°C. Then [12~I]IGF II, 40,000 cpm in 3 ]~1 was added and incuba t ed for ano the r 30 rain a t 4°C. PMP-BSA, 200 ng in 8 I11 was added and the r eac t i on mix ture incuba ted for 60 min. In cont ro ls r ecep to r or PMP-BSA was omit ted. The PMP-BSA was immunoprec ip i t a t ed by i ncuba t ing a t room t e m p e r a t u r e for 30 min and for 16 h a t 4°C with 60 ~g of IgG aga in s t PMP-BSA. Controls r e c e i v e d 6O lag preimmune IgG. The immunoprec ip i t a t e s were c e n t r i f u g a t e d , washed twice with buf fe r A, and a s sayed for r a d i o a c t i v i t y .
De te rmina t ion of i nh ib i t i on c o n s t a n t s of PMP and P M P - l y s - a p r o t i n i n for PMP- BSA binding to f ib rob las t s : The b inding of [t2~I]PMP-BSA to f ib rob las t s was car r ied out a t 4°C as descr ibed (7). The KD for PMP-BSA was de te rmined us ing 0.125 - 2 x 10 -l° PMP-BSA. The compe t i t i ve i nh ib i t i on of b inding of PMP-BSA by PMP or PMP- iys -ap ro t in i .n was de te rmined a t 6 x 10 -6 and 5 x 10 -8 M of t he inhibi~ors.
Results
Aftrlnity labelling of the M6PIIGF II receptor with,, ~6F-iigand: Aprotinin, the
basic pancreatic trypsin inhibitor was chosen as accep~tor potypeptide for PMP.
12~9
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A p r o t i n i n h a s a m o l e c u l a r w e i g h t o f 6500 , 5 a m i n o g r o u p s a s a c c e p t o r s i t e s f o r
t h e PMP a n d 4 t y r o s i n e r e s i d u e s , w h i c h c a n be i o d i n a t e d (12) . T h e PMP-
s u b s t i t u t e d a p r o t i n i n c o n t a i n e d 3 .9 m o l e s PMP p e r tool a p r o t i n i n s u g g e s t i n g t h a t
4 o u t of 5 a m i n o g r o u p s a v a i l a b l e (12) a r e c o u p l e d w i t h PMP. To i n c r e a s e t h e
p r o b a b i l i t y of c r o s s l i n k i n g , t h e c a r b o x y l i c g r o u p s of t h e P M P - a p r o t i n i n we re
s u b s t i t u t e d w i t h l y s y l - t r i p e p t i d e s . T h e r e s u l t i n g P M P - l y s - a p r o t i n i n h a d a n
a p p a r e n t s i z e of 12 ,500 . I t s a f f i n i t y to M 6 P - r e c e p t o r s w a s i n t e r m e d i a t e b e t w e e n
t h a t of PMP a n d PMP-BSA. T h e PMP-BSA b o u n d to M6P/IGF II r e c e p t o r s a t t h e
ce l l s u r f a c e of f i b r o b l a s t s w i t h a KD of 2.6 x 10-~°M. P M P - l y s - a p r o t i n i n a n d
PMP i n h i b i t e d t h e b i n d i n g o f PMP-BSA w i t h Kl 's o f 3 .6 x 10-#M a n d 4 .3 x
10-6M, r e s p e c t i v e l y .
F o r a f f i n i t y l a b e l l i n g p u r i f i e d M6P/IGF II r e c e p t o r was i n c u b a t e d w i t h
[ ~ 2 ~ I ] P M P - l y s - a p r o t i n i n a n d c r o s s l i n k e d w i t h DSS. [ l z s I ] P M P - l y s - a p r o t i n i n was
c r o s s l i n k e d to t h e r e c e p t o r a s wel t a s to m a t e r i a l of h i g h e r a n d l o w e r m o l e c u l a r
w e i g h t (Fig. 1, l a n e 1). Cross l i n k i n g to t h e r e c e p t o r a n d to t h e h i g h e r
m o l e c u l a r w e i g h t m a t e r i a l was i n h i b i t e d by M6P (Fig. 1, l a n e 2 a n d 5), b u t n o t
b y g l u c o s e 6 - p h o s p h a t e (Fig. 1, l a n e 6). T h e h i g h e r m o l e c u l a r w e i g h t m a t e r i a l i s
l i k e l y to r e p r e s e n t c r o s s l i n k e d a g g r e g a t e s of t h e r e c e p t o r . T h e c r o s s l i n k i n g to
t h e l o w e r m o l e c u l a r w e i g h t m a t e r i a l was n o t a f f e c t e d by M6P (Fig. 1, l a n e 2) b u t
r e d u c e d in t h e p r e s e n c e of 5 mM l y s i n e (Fig, 1, l a n e 4 - 6 ) . C ros s l i n k i n g to t h e
lowe~" m o l e c u l a r w e i g h t m a t e r i a l was a l s o s e e n w h e n no r e c e p t o r was p r e s e n t i n
t h e i n c u b a t i o n m i x t u r e (Fig. 1, l a n e 3), or w h e n t h e r e c e p t o r a n t i b o d i e s we re
m
1 2 3 4 5 6
Fig. 1: Cross l inking of [ lzsI ]PMP-lys-aprot in in to M6P/IGF II receptor .
M6P/IGF II recep tor was i ncuba t ed wi th [ t~sI]PMP-lys-aprot inin and cross l inked with 0.06 mM (lane 1-3) or 0.6 M DSS ( lane 4 -6 ) . The incuba t ion mix tures of lane 4 - 6 con ta ined 5 mM lysine. The addi t ions and v a r i a t i o n s were as follows: l ane 1 and 4: none; lane 2 and 5 : 5 mM mannose 6 - p h o s p h a t e ; lane 3: omission of receptor ; l ane 6 : 5 mM glucose 6 - p h o s p h a t e . Labelled M6P/IGF II r ecep to r (-~) as well as labe l led mate r ia l of h igher (~) and lower ( - ) molecular weight mate r ia l are indica ted . The r ad ioac t ive mater ia l a t t he bottom r e p r e s e n t s f ree [125I]PMP-lys-aprotinin.
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r e p l a c e d b y p r e i m m u n e Ig ( n o t s h o w n ) . ~'q b e l i e v e t h a t t h e l o w e r m o l e c u l a r
w e i g h t m a t e r i a l r e p r e s e n t s c r o s s l i n k e d P M P - t y s - a p r o t i n i n t h a t b i n d s
u n s p e c i f i c a l l y to t h e i m m u n o s o r b e n t . L y s i n e m a y r e d u c e t h e s e l f - a g g r e g a t i o n of
P M P - l y s - a p r o t i n i n a n d t h e r e b y d e c r e a s e t h e c r o s s l i n k i n g .
A f f i n i t y l a b e l l i n g of t r y p t i c r e c e p t o r f r a g m e n t s w i t h [ l ~ I ] P M P - l y s - a p r o t i n i n
a n d [~aI]IGF II: I n c u b a t i o n of t h e p u r i f i e d r e c e p t o r f o r 12 h w i t h t r y p s i n
p r o d u c e d 6 m a j o r i m m u n o r e a c t i v e f r a g m e n t s of t h e r e c e p t o r w i t h s i z e s b e t w e e n
23 a n d 37 .5 kDA (Fig. 2, l a n e 2). T h e p a t t e r n of t h e s e f r a g m e n t s r e m a i n e d
c o n s t a n t i f t h e d i g e s t i o n w a s e x t e n d e d fo r 24 h. A f f i n i t y l a b e l l i n g of t h e
r e c e p t o r f r a g m e n t s w i t h [ 1 2 a I ] P M P - l y s - a p r o t i n i n y i e l d e d f o u r m a j o r p r o d u c t s of
27 .5 - 39 .5 kDa (Fig. 3, l a n e 1). L a b e l l i n g of t h e s e f r a g m e n t s w as i n h i b i t e d b y
M6P (Fig. 3, l a n e 2) . T h e a m o u n t of [ l = s I ] P M P - l y s - a p r o t i n i n c r o s s l i n k e d to
t h e s e f r a g m e n t s was 12 % of t h a t c r o s s l i n k e d to t h e i n t a c t r e c e p t o r (Fig. 3,
1
1 2 3 4 5 6
37 .5
33 .5
29 .5 27 .5
25 .0 23 .0
39.5
33.5 3O 27.5
- - 37.5
- - 28
Fig. 2: Western blot of t ryp t i c f ragments of the M6P/IGF II recep tor
- - 21
- - 19
Lane 1: S ta in ing of M6P/IGF II r ecep to r (-~) with s i l v e r
Lane 2: Trypt ic f ragments of the M6P/IGF II recep tor were s epa ra t ed by SDS- polyacrylamid gel e lec t rophores i s , t r a n s f e r r e d to n i t roce l lu l a r e and immunos ta ined with recep tor an t ibod ies . The molecular weights (kDa) of the recep tor f ragments are indica ted .
Fig. 3: Cross l ink ing of [ lzaI]PMP-lys-aprot in in and [~=sIIIGF II to t ryp t i c r ecep to r f ragments and M6P/IGF II recep tor
Trypt ic r ecep to r f ragments ( lane 1, 2) and M6P/IGF II recep tor ( lane 5) were i n c u b a t e d wi th [ l=~l]PMP-lys-aprot inin and cross l inked wi th 0,06 mM DSS. Similarly, t r yp t i c recep tor f r agments ( lane 3 and 4) and M6P/IGF II r ecep to r ( lane 6) were i n c u b a t e d with [lznI]IGF II and cross l inked wi th 0.6 mM DSS. The i n c u b a t i o n mixture of lane 2 con t a ined 5 mM mannose 6 - p h o s p h a t e , t h a t of l ane 4 an excess of un labe l l ed IGF I and IGF II ( p r epa ra t i on 1932). The pos i t ion of the labe l led recep tor (-~) and the size (kDa) of the labe l led t ryp t i c recep tor f r agments are ind ica ted .
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Table 1 Coprec ip i ta t ion of [12~I]IGF II recep tor complexes and PMP-BSA receptor
complexes by an t ibod ies aga ins t PMP-BSA
Var ia t ion of s t a n d a r d assay* Immunoprecip i ta ted [IuaI]IGF II (cpm)
2060 minus recep tor 38 minus PMP-BSA 162 preimmune Ig in s tead of recep tor an t ibod ies 101 plus un labe l l ed IGF II (4 lag) 527
*For s t a n d a r d as say see Mater ia ls and Methods
l a n e 5). [12~I]IGF It was c r o s s l i n k e d to two m a j o r (28 a n d 37 ,5 kDa) a n d two
m i n o r (19 a n d 21 kDa) r e c e p t o r f r a g m e n t s (Fig. 3, l a n e 3). C ros s l i n k i n g to t h e s e
f r a g m e n t s was i n h i b i t e d b y a n e x c e s s of u n l a b e l l e d IGF II (Fig. 3, l a n e 4). T h e
a m o u n t o f [12aI]IGF II c r o s s l i n k e d to f r a g m e n t s was 26 % of t h a t c r o s s l i n k e d
to t h e i n t a c t r e c e p t o r (Fig. 3, l a n e 6).
B i n d i n g o f PMP-BSA a n d IGF II to t h e s a m e r e c e p t o r : P u r i f i e d h u m a n l i v e r
M6P/IGF II r e c e p t o r was i n c u b a t e d w i t h [1251]IGF II a n d PMP-BSA. A n t i b o d i e s
a g a i n s t t h e PMP-BSA w e r e a d d e d to t h e r e a c t i o n m i x t u r e s . F i v e % of t h e a d d e d
[Iz~I]IGF II w e r e r e c o v e r e d in t h e i m m u n o p r e c i p i t a t e ( T a b l e 1). O m i s s i o n of
r e c e p t o r or PMP-BSA or r e p l a c e m e n t of t h e PMP-BSA a n t i b o d i e s b y p r e i m m u n e Ig
d e c r e a s e d t h e ~ S s o c i a t i o n of t h e [12~I]IGF II w i t h t h e i m m u n o p r e c i p i t a t e b y
> 83 % ( T a b l e 1)., T h i s i n d i c a t e s t h a t t h e a s s o c i a t i o n of t h e IGF t i w i t h t h e
i m m u n o p r e c i p i t a t e d e p e n d e d on t h e p r e s e n c e of t h e r e c e p t o r , t h e PMP-BSA a n d
t h e PMP-BSA a n t i b o d i e s . T h e s p e c i f i c i t y of t h e b i n d i n g of [lzsI]IGF II to t h e
PMP-BSA r e c e p t o r c o m p l e x e s was i n d i c a t e d b y t h e i n h i b i t o r y e f f e c t of u n l a b e l l e d
IGF II ( T a b l e 1). T h e s e r e s u l t s p r o v i d e e v i d e n c e t h a t p u r i f i e d M6P/IGF II
r e c e p t o r c a n b i n d s i m u l t a n e o u s l y PMP-BSA a n d IGF II.
Discussion
P M P - l y s - a p r o t i n i n w as f o u n d to be a s u i t a b l e l i g a n d fo r a f f i n i t y l a b e l l i n g of
t h e M6P/IGF II r e c e p t o r b y c r o s s l i n k i n g . I t s a f f i n i t y (~ 10 -7 - 10-SM) i s
i n t e r m e d i a t e b e t w e e n t h a t of PMF-BSA a n d l y s o s o m a l e n z y m e s (= 10 -~ -
10 -1o f¢~) a n d PMP (= 10 -n - 10-6M). I t s low m o l e c u l a r w e i g h t p e r m i t t e d a n
i n i t i a l c h a r a c t e r i z a t i o n of M6P/IGF II r e c e p t o r w i t h M6P b i n d i n g a c t i v i t y . T h e
t r y p t i c r e c e p t o r f r a g m e n t s t h a t w e r e a f f i n i t y l a b e l l e d b y [ 1 2 ~ I l P M P - l y s - a p r o t i n i n
w e r e d i f f e r e n t f rom t h o s e l a b e l l e d b y [IzsI]IGF II. T h i s s u p p o r t s t h e v i e w t h a t
t h e M6P a n d IGF II b i n d i n g s i t e s r e s i d e in d i f f e r e n t p a r t s of t h e r e c e p t o r . A
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s imi lar conc lus ions was drawn from the o b s e r v a t i o n t h a t the two types of
l igands do not compete for b inding (3, 5) and t h a t the two types of b inding
s i t e s are blocked by d i f f e r e n t r ecep to r a n t i s e r a (5).
The p r e sen t r e s u l t s do not allow prec ise e s t i m a t e s of the molecu la r weights
of the f r agments wi th M6P or IGF II b inding a c t i v i t y . Cross l inking of t he two
l igands, namely t h a t of the neog lycopro te in [125I]PMP-lys-aprotinin may cause
anomalous b e h a v i o u r in SDS-po lyac ry lamide gel e l ec t rophore s i s . If the a p p a r e n t
s ize of the l abe l l ed f ragments is co r rec ted for the s ize of the l igands , 12.5 kDa
for P M P - l y s - a p r o t i n i n and 6.5 kDa for IGF II, and if one assumes a
s t oech iome t ry of 1:1 be tween the f ragments and the l igands, the M6P binding
r ecep to r f r agments h a v e s izes of 15 - 27 kDa and the IGF II b inding f ragments
h a v e s izes of 11.5 - 31.5 kDa. The e x t r a c y t o s o l i c por t ion of M6P/IGF tI r e c e p t o r
is made up of 15 homologous r e p e a t s of 134 - 167 amino acids (1, 2). The
s imi l a r i t y be tween the s izes of the r e p e a t s and the r ecep to r f ragments wi th M6P
or IGF II b inding s i t es sugges t s t h a t the s t r u c t u r e s n e c e s s a r y for b inding e i t h e r
type of l igand are con ta ined wi th in one or - a t maximum - wi th in two
ne ighbour ing r epea t s . The fol lowing two poss ib i l i t i e s , however , should be
cons idered . It is poss ib le t h a t two or more r ecep to r f r agments make up the M6P
and /o r the IGF II b inding s i t e s and become cross l inked by DSS to each o the r or
to the l igand. We could exclude , however , t h a t cross l ink ing of the r ecep to r
f r agments in the absence of l igands induces a s ize sh i f t of the immunoreac t ive
r ecep to r f r agments (not shown). Fu r the r , the s ize of the M6P binding f r agmen t s
may be o v e r e s t i m a t e d , s ince P M P - l y s - a p r o t i n i n as a m u l t i v a l e n t l igand may
become cross l inked to more t h a n one M6P binding r ecep to r f ragment .
P rev ious s t ud i e s h a v e shown t h a t pur i f i ed M6P/IGF II r ecep to r p r e p a r a t i o n s
bind both types of l igands. It was not c lear , w he the r the two types of l igands
bind to subc l a s se s of the recep tor , which are copur i f ied , or to the same
receptor . We were able to show t h a t M6P/IGF II r ecep to r s form complexes wi th
IGF II and PMP-BSA t h a t can be immunoprec ip i t a t ed with an t ibod ies aga in s t
PMP-BSA. This c l ea r ly demons t r a t e s t h a t a s ingle r e c e p t o r is able to bind M6P
and IGF II. At p r e s e n t i t is not known whe the r the M6P/IGF II r e cep to r t h a t
bind l igands are of monomeric or ol igomeric s t r uc tu r e .
In summary, the p r e s e n t r e s u l t s p rov ide ev idence t h a t the M6P and IGF II
binding s i t e s a re loca ted a t d i f f e r e n t pa r t s of the M6P/IGF II r ecep to r and t h a t
a s ingle r ecep to r can bind both t ypes of l lgands s imul t aneous ly .
Acknowledgements
We t h a n k Dr. R.E. Humbel, Ztirich, for the gi f t of IGF II and a mix ture of IGF I/IGF II. This s t udy was suppor t ed by the Deutsche F o r s c h u n g s g e m e i n s c h a f t (SFB 236) and the Fonds der Chemischen Indus t r i e .
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Vol, 152, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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