Retinal insulin receptors. 1. Structural heterogeneity and functional characterization

Exp. Eye Res. (1987) 45, 823-835

R e t i n a l I n s u l i n R e c e p t o r s . 1. S t r u c t u r a l H e t e r o g e n e i t y a n d Functional Characterization R. J. WALDBILLXG*~, R. THEODORE FLETCHER*, GERALD J. CHADER*, SANKARAN RAJAGOPALAN~, MERLYN RODRIGUES~ AND D. LEROITIt§

* Laboratory of Retinal Cell and Molecular Biology, ~ Laboratory of Pathology, National Eye Institute, National Institutes of Health, Bethesda, MD, § Diabetes Branch, National' Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, M D 20892, U.S.A. (Received 6 February 1987 and accepted 24 M a y 1987) Neural cells of the bovine retina contain specific, high-affinity receptors for insulin. When solubilized and wheat-germ purified, these receptors exhibit a kinase activity that is capable of phosphorylating the receptor's fl-subunit (autophosphorylation) and a tyrosine-containing exogenous substrate, poly (Glu. Tyr) 4: 1. Studies of the structure of retinal insulin receptors revealed the existence of two insulin receptor subpopulations. For these populations, the apparent molecular weights of the a-subunit were 120- and 133 kDa. This structural heterogeneity does not appear to be related to the presence of vascular contamination and stands in contrast to the brain and liver where a single a-subunit type was found (120 kDa for brain and 133 kDa tbr liver). In addition to being distinguishable by their molecular weights, the two populations of retinal insulin receptors could be distinguished in terms of (a) their solubility in Triton X-100, (b) glycosy|ation, and (c) recognition by anti-insulin rec~eptor antibody. Despite these structural differences, the two t~pulations of retinal insulin receptors appear to have similar insulin binding affinities. ]x*ey words: insulin receptors; retina; autophosphorylation ; tyrosine kinase.

1. I n t r o d u c t i o n Insulin receptors are membrane-bound glycoproteins consisting of subunits with e x t r a c e l l u l a r a n d c y t o p l a s m i c d o m a i n s ( E b i n a e t al., 1985; U l h ' i c h e t al., 1985). I t h a s been shown t h a t insulin binding, to t h e a - s ~ b u n i t s t i m u l a t e s tyrosine-specific p h o s p h o r y l a t i o n o f t h e / ? - s u b u n i t as well as e x o g e n o u s s u b s t r a t e s (Zick, K a s u g a , K a h n a n d R o t h , 1983; Z i c k , G r u n b e r g e r , R e e s - J o n e s a n d Coral, 1985). O v e r t h e p a s t f e w y e a r s , s u c h r e c e p t o r s h a v e b e e n d e m o n s t r a t e d in a v a r i e t y o f t i s s u e s a n d cell t y p e s (Kasuga, Akanuma, I w a m o t 0 a n d K o s a k a , 1978; L e M a r c h a n d e t al., 1976; T a k a y a m a , W h i t e , L a u r i s a n d K a h n , 1984). A l t h o u g h t h e t r a d i t i o n a l v i e w is t h a t n e u r a l t i s s u e is i n s e n s i t i v e t o i n s u l i n , r e c e n t w o r k h a s s h o w n t h a t i n s u l i n r e c e p t o r s a r e p r e s e n t in b o t h t h e c e n t r a l ( H a v r a n k o v a , R o t h a n d B r o w n s t e i n , 1978; P a c o l d a n d B l a c k a r d , 1979) a n d p e r i p h e r a l ( W a l d b i l l i g a n d L e R o i t h , 1987) n e r v o u s s y s t e m . I n t e r e s t i n g l y , t h e b r a i n i n s u l i n r e c e p t o r h a s b e e n s h o w n t o be a p p r o x i m a t e l y I 0 k D a s m a l l e r t h a n i t s c o u n t e r p a r t in p e r i p h e r a l n e u r a l a n d n o n - n e u r a l t i s s u e s ( I : l e i d e n r e i c h , Z a h n i s e r , B e r h a n u , B r a n d e n b u r g a n d O l e f s k y , 1983; Y i p , M o u l e a n d Y e u n g , 1980; H e n d r i c k s , A g a r d h , T a y l o r a n d R o t h , 1984; C i a r a l d i , R o b b i n s , L e i d y , T h a m m a n d ¢ To whom all correspondence should be addressed, at Building 6A, Room BIA09, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A. .4 bbreviations : WGA : wheat germ agglutinin ; PMSF : phenylmethylsuifonyl fluoride ; IGF: insulinlike growth factor; K R P : Krebs-Ringer phosphate; PEG: polyethylene glycol; BSA: bovine serum albumin; EDTA: ethylene diaminetatraacetic acid; ATP: adenosine 5"-triphosphate; CTP: cytidine 5"-triphosphate; MES: 2(N-morpholino)-ethanesulfonic acid. 0014-4835/87/120823 + 13 $03.00/0

© 1987 Academic Press Limited

824

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B e r h a n u , 1985; L o w e and L e R o i t h , 1986; S i m o n a n d L e R o i t h , 1986; S h e m e r , P e n h o s a n d R o t h , 1986; Waldbillig and L e R o i t h , 1987). A d d i t i o n a l l y , g l y c o s y l a t i o n o f t h e brain insulin r e c e p t o r is k n o w n t o be d i f f e r e n t f r o m t h a t f o u n d in receptor'J in liver a n d ~ d i p o c y t e s ( H e i d e n r e i c h e t al., 1983; L o w e a n d L e R o i t h , 1986). A d d i t i o n a l l y , g l y c o s y l a t i o n o f t h e b r a i n insulin r e c e p t o r is k n o w n t o be d i f f e r e n t fi'om t h a t f o u n d in r e c e p t o r s in liver a n d a d i p o c y t e s ( H e i d e n r e i c h e t al., 1983; L o w e a n d L e R o i t h , 1986). T h e r e t i n a is a n integral, albeit specialized p a r t o f t h e central n e r v o u s s y s t e m (CNS) a n d is also k n o w n t o c o n t a i n insulin r e c e p t o r s ( H a v r a n k o v a , R o t h a n d B r o w n s t e i n , 1978). A l t h o u g h insulin m a y p l a y a role in r e t i n a l glucose m e t a b o l i s m , it m a y also h a v e o t h e r ' n o n - c l a s s i c a l ' effects, e.g. in t h e d e v e l o p m e n t of r e t i n a l s y n a p s e s ( P u r o and A g a r d h , 1984). W e t h u s felt it o f intere,~t to m o r e fully define t h e n a t u r e o f insulin r e c e p t o r s in t h e r e t i n a as to t y p e a n d specific c h a r a c t e r i s t i c s .

2. M a t e r i a l s a n d M e t h o d s Mcaerials Monoiodinated [125I]I)orcine insulin purified by HPLC (radio-receptor grade, specific activity 25(}-370 mCi Ing -t) and Ia"P]ATP (1000-3000 Ci mmol -l) were purchased fi'om New England Nuclear (Bast, on. MA). Porcine insutin was purchased f~om Elanco (Indianapolis, IN). S y n t h e t i c human l)roinsulin (A-18-4U6-253) was a gift from l)r Bruce Frank. Eli Lilly & Co. (Indianapolis, IN). Bovine serum albumin (fraction V, HPLC insulin free) was lmrchased from Armor Pharmaceutical Co. (Kankakee, ILL Wheat germ agglutinin ((]iyeamino-silex, ~,VGA) was purchased from Miles Yeda Ltd (Rehovot, Israel). The synthetic tyrosine-containing polypeptide poly (Glu, Tyr) 4:1 and neuraminidase (from Cto,~tridium perfrinflen~s tyl)e X) were l)urchased from Sigma Chemical Co. (St Louis, MO). All other chemicals used were reagent grade.

Preparation of liB,sue Bovine eyes, brain and liver were obtained fi'om steers killed by exsanguination at a commercial slaughterhouse. Eyes were stored overnight on ice, in i~ae dark. Retinas were then separated from the rest of"the eye and frozen a t -- 70°(; until use. Brain and liver tissues were r~moved witilin 20-40 rain of exsanguination, placed in watertight plastic bags and packed in ice for" transportation to the laboratory. Brain and liver" crude membranes were prepared immediately ut)on arrival. For some retinas, crude membranes were prepared immediately upon arrival at the laboratory.

Preparation of crude plaama membranes Crude membranes were l)rel)ared by differential centrifugation (Havrankova, Roth and Brownstein, 1978). After homogenization on ice in a 1 m,~t sodium carbonate buffer containing Im,~1 phenyhnethylsulfonyl fluoride (PMSF) and 10/,m m1-1 leupeptin as l)rotease inhibitors, the homogenates were centrifuged at 600g for 15 rain at 4°C. The s,Jpernatant obtained from this centrifugation was recentrifuged at 2 0 0 0 0 g for 30 min. These pellets were resuspended in fresh buffer and centrifuged again a t 20000 g for 30 rain. Finally the pellets were resuspended in K r e b s - R i n g e r phosphate buffer (KRP, without calcium, pH 8"0). In a separate series of experiments designed to detect potential vascular contamination of retinal membranes, the tissue homogenates were centrifuged a t 600-, 2000-, 5000-, or 10000g for 15 rain at 4°C. The sutmrnate obtained from this centrifugation was recentrifuged a t 2 0 0 0 0 g for 30 rain. These pellets were resuspended in K r e b s - R i n g e r phosphate buffer without calcium (pH 8"0).

Factor V I I I analysis To determine whether vascular tissue was contaminating retinal crude m e m b r a n e preparations, centrifugation fractions of whole retina homogenate were examined for

INSULIN

RECEPTORS

IN B O V I N E

RETINA

825

factor V H I antigen. M e m b r a n e s in t h e v a r i o u s fractions were fixed in a c e t o n e for I0 rain, frozen and c u t into s e c t i o n s 4 ttm thick. P r i m a r y polyelonal a n t i b o d y a g a i n s t the(or V I I I related antigen (Accurate Chemical Co., W e s t b u r y , NY) was diluted 1:20 in phosphatebuffered saline (PBS), p H 7"4, prior to use. Followi~g i n c u b a t i o n in a m o i s t c h a m b e r for I hr a t room temperature, t h e a n t i b o d y was rinsed off and replaced with fluoresce!n-labeled g o a t a n t i - r a b b i t lgG a t a dilution o f ! :20, applied for 30 min. then washed in buffer before mounting.

Preparatio~* of purified reccptor,~ M e m b r a n e s (5-6 mg protein ,nl-' in 50 mM Hepes, 150 ,n~ NaCI buffer, pH 7"6), were solubilized on ice for 3 hr using i % Triton X-100 in the presence of ! m~! P M S F . The mixture was centrifuged at ! 30000 g for 45 rain a t 4°C and the s u p e r n a t a n t applied three successive times to a 2-ml W G A - a g a r o s e cohnnn. T h e column was washed with 40 ml buffer containing 150 m.~ sodium clfloride. 0"1 ~¼~ Triton X-100, 50 m,~ mM H e p e s (pH 7"6) and the adsorbed material elutetl in l-ml fi'actions using 0"3 ,~ N-aeetyl glucosamine in the s a m e buffer (Hedo. Harrison and Roth. 1981). Protein concentration o f the e l u a t e s was assessed with the Bio R a d a s s a y using bovine s e r u m a l b u m i n (BSA) (iiluted in 50 trim H e p e s containing 151)m,',l sodium chloride (pH 7"6), 0"1 ~¼~ Triton X-100, and 0'3 ,xl N - a e e t y l glucosamine as protein s t a n d a r d (Bradford. 1976)~ (Bio R a d Assay, Richmond, CA).

ln.sulin binding ,studie,~" Insulin binding to crude m e m b r a n e s was measured in calcium-free KR.P buffer (pH 8"0) using [12~l]insulin (0-03 n~1) a n d various concentrations of unlabeled porcine insulin and h u m a n proinsulin. This buffer also contained 1 % BSA and i m g m1-1 baeitracin. ~Iembranes were used a t a final protein c o n c e n t r a t i o n of 1.5-2"0 mg ml -t. Incubation was conducted o v e r n i g h t a t 4°C and was sti~pped b y a d d i n g 50/~1 of 0"5 m ice-cold s~erose and c(mtritk~ging (! 2 0 0 0 g ; 4¢C) for 8 rain. T h e surface o f the pellet was washed with chilled K R P buffer and recentrifuged for 4 rain. D e g r a d a t i o n of the tracer w~ts e s t i m a t e d as non-precipitable material using 5"/0 trichloroacetie acid and was fi)und to he less than 4 % . Insulin binding to wheat=germ purified receptors (He(lo, H a r r i ~ m a n d Roth, '!981 ; Simon and l , e R o i t h . 1986) was s t u d i e d l)y incubating 25/~1 o f l)urified receptors witi~ 100/ll [~a'~l]insulin (0"03 nM). 25 td o f 50 mM H e p e s - 1 5 0 m,xi NaCl ( p H 8"0) o v e r n i g h t at 4°C in t h e presence of 5 0 / d of various c o n c e n t r a t i o n s of unlabeled l)orcine insulin or h u m a n t)roinsulin. The final incubation v o l u m e ( 2 0 0 / d ) also contained I ~¼~BSA a n d I'0 mg m1-1 bacitraein. To t e r m i n a t e t h e incut)ation 100/~l ¢)f 0"3% bovine gamma-glol)ulin and 30(~/d of 2 5 % p o l y e t h y l e n e glycol ( P E G ) were a d d e d to t h e tut)es and centrifuged a t 2 5 0 0 g fi)r 15 rain at 4°C. T h e pellet was washed once with 300 t d o f 12"5'¼) P E G ; t h e s u p e r n a t a n t xvau aspirated and the radioactivity in t h e pellet c o u n t e d in a g a m m a - c o u n t e r .

Cro.~linki~g of [l~sIJln,~ulin to crude 71~embranea" Crosslinking of [12~l]insulin to crude m e m b r a n e s was p e r f o r m e d as previously, described (Pi.l~.h a n d Czech, 1980) with m i n o r modifications. In different e x p e r i m e n t s , t h e concentration of [12sl]insulin used varied from 3"0- to 0.03 nM. Memhranes-[5 mg protein (I-5 ml) -I] were i n c u b a t e d with [~al]insulin o v e r n i g h t a t 4°C in a calcium-free K R P buffer containing I % BSA, I mg ml -~ baeitracin a n d v a r y i n g concentrations o f unlabeled insulin or insulin-like g r o w t h factor-I (IGF I ) . T h e binding reaction was s t o p p e d b y eentrifugation a t 12000 g for 30 rain a t 4°C. The resulting pellet was washed twice in ice-cold KFCP bufibr (pH 8"0) ilr the absence of BSA and r e s u s p e n d e d in 1"5 ml K R P b'uffer w i t h o u t BSA (pH 8"0). Crosslinking o f the b o u n d [l~aIJinsulin t o t h e receptors was performed lay t h e addition of 15 t d of 10 m.~l disuccinimidyl s u b e r a t e (DSS) tbr 15 rain on ice. The erosslinking reaction was t e r m i n a t e d ' b y a d d i n g 300 td of 100 lnM T r i s - 1 0 mM E D T A (pH 7"8). W h e n t h e glycosylation features of the insulin receptors were to be e x a m i n e d ' the erosslinked material was treated with n e u r a m i n i d a s e (type 10; 2"5 U ml -~ in 5.0 m~l M E S - I - 0 re.x1 CaCl2) tor 15 min at 37°C. W h e n the crosslinked r e c e p t o r s were to be a p p l i e d directly t o S D S - P A G E gels, the crosslinked material was c e n t r i f u g e d ( I 2 0 0 0 g , 4°C, 15 rain) a n d the pellet resuspended in 6 0 - 1 6 0 t d o f sample buffer ( 2 % SDS, 5 % ~q-mereaptoethanol. 10"0% glycerol. 0"01% b r o m o p h e n o l blue, 40 mM Hepes, p H 7"6). T h e resu~l)~nded material was then boiled (10

826

R. J. W A L D B f L L I G ET AL.

rain), centrifuged (12000 g, 15 min) and 50/,l of the s u p e r n a t a n t applied to the gel. When the crosslinked material was to be immunoprecipitated, the erosslinked material was centrifuged (12000g, 4°C, 15 min) and the pellet resuspended in 1-0 mi 50 mM Hepes-150 mM NaCi (pH 7"6) containing 100/Ll of l0 % Triton X-100. Following an overnight solubilization (4°C, with continuous inversion), the tubes were c e n t r i f u g e d at 130000g for 45 rain (4°C). Following separation from the pellet, 10/zl of anti-insulin receptor antisera (B 10, a generous gift of Dr Simeon I. Taylor, Bethesda, MD) was added to the s u p e r n a t a n t and the solution mixed by continuous inversion (overnight, 4°C). The r e c e p t o r - a n t i b o d y complex was immunoprecipitkted with 200/LI of Pansorbin as previously described (Lowc and LeRoith, 1986). The immunoprecipitate was boiled in 60-160/11 of sample buffer for I0 rain and then centrifuged at. 12000 g for 15 min. Samples (50/d) of the supernate were analysed using 0" 1% S D S - P A G E and autoradiography (Pilch and Czech, 1980).

Crosslinking of [l~Sl]in~ulin to WGA-purified receptors To determine the a p p a r e n t molecular weight of the solubiliT.ed and WGA-purified reccptor's binding subunit, I00/*l of the purified receptor preparation was incubated with 25/tl of [12hi]insulin (final concentration 3 " 0 h i ) , 25tll of unlabeled insulin (final concentration 10-6 M) or 25/L1 50"0 mM Hepes-150 mM NaCl overnight at 4°C. Following binding, insulin was crosslinked to the bindifig site using 3/~.l of 50 m i disuccinimidyl suberate at 4°C for 45 rain. The crosslinking reactio)z wa.~ terminated by adding 38-3/l] of 5 × sample buffer. The crosslinked material was boiled, centrifuged (12000 g fi)r 15 rain) and 50 t t] samples were run on an S D S - P A G E gel and exposed to film. Autoradiography was used to localize the bands.

Autophosphorylation of the in,ulin receptor/~-,ubunit Phosphorylation of the/]-subunit of retinal insulin receptors was performed at 23°C under conditions previously described (Zick, Kasuga, Kahn and Roth, 1983; Rees-Jones, Hendricks, Quarum and Roth, 1984). Purified receptors were incubated in the absence and presence of 10-~- and 10 -7 M insulin for 30 rain at 23°C. The phosphorylation reaction was initiated by the addition ofT,[a2P]ATP, Mg 2÷ (20 mM final concentration), Mn 2. (3"0 raM), CTP (! "0 m,~l), A T P (50/ZM) and v a n a d a t e (1"0 mM). Ten rain later the reaction was terminated by the addition of stopping solution (Triton X-100, 0"4%; EDTA, 20 raM; sodium fluoride, 200 m,~ : disodium pyrophosphate, 40 mM; sodium phosphate, 40 mM ; ATP, 40 m.~z; Hepes, 40 m M) and samples were run on S D S - P A G E with or without prior immunoprecipitation by anti-insulin receptor antiserum (B 10) as previously described (McElduff, Schroer and Taylor, 1984).

Phosphorylation of exogenous substrate To evaluate the kinase a c t i v i t y ' o f retinal insulin receptors for exogenous substrate, the incorporation of a2p into poly (Glu, Tyr) 4 : 1 was monitored. The final concentration of this substrate was 50 mM and the assay was performed under conditions previously described (Zick, Kasuga, K a h n and Roth, 1983). Receptors were preincubated with insulin (10 -°- and 10-:M) for 3 0 m i n at room t e m p e r a t u r e prior to the addition of the 7,-labeled [a2P]ATP mixture in the absence o f M n ~+and vanadate. The aup incorporation into exogenous substrate was d e t e r m i n e d with a beta-counter. 3. R e s u l t s

Competition inhbition of [125I]insulin-specific binding to crude membranes [z25I]Insulin b i n d i n g to r e t i n a l c r u d e m e m b r a n e p r e p a r a t i o n s was p e r f o r m e d o v e r n i g h t a t 4°C a t a final p r o t e i n c o n c e n t r a t i o n o f 2 m g ml -z. T h e specific b i n d i n g o f [z25I]insulin wagr9"4%. P o r c i n e insulin d i s p l a c e d t h e [z2sI]insulin w i t h a h a l f - m a x i m a l v a l u e a t 0"5 riM, w h e r e a s h u m a n p r o i n s u l i n h a l f - m a x i m a l d i s p l a c e m e n t was a t 100 nM (.Fig. I). F o r c o m p a r i s o n , [125I]insulin b i n d i n g to b o v i n e b r a i n a n d liver was 2-0- a n d

INSULIN RECEPTORS

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IN B O V I N E R E T I N A

827

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Proinsulin

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UNLABELLED HORMONE (nM)

Fro. I. Bovine retinal crude membranes: competition-inhibition of specific [l=bI]insulin binding. Crude membranes from retina were incubated overnight (4°C) with [l=~I]insulin (0"03 riM) in the presence or absence of various concentrations of unlabeled porcine insulin or human proinsulin. Initial specific binding (total binding minus non-specific binding) in the absence of unlabeled hormone was 9.4 %. Nonspecific binding (binding in the presence of a large excess (i0 -e M) of unlabeled insulin) was 20.2 % of the total binding. IC~0 indicates the concentration of unlabeled hormone that produced a half-maximal inhibition of specific binding, 55 50 45

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W G A Fractions

FIe. 2. WGA purification of solubilized bovine retina. Crude membranes from bovine retina were solubilized in l °Z~ Triton X-100 and applied to a WGA-agarose column. Specific [12hi]insulin binding (percentage of the added counts t h a t specifically bound) was measured in receptors eluted from the column with N-acetyl-glucosamine (0"3 r,l).

3"0 % a t 1-0 m g p r o t e i n m l -z. F o r t h e s e t i s s u e s , t h e h a l f - m a x i m a l d i s p l a c e m e n t w i t h u n l a b e l e d i n s u l i n w a s 3"0- a n d 5-0 nM r e s p e c t i v e l y ( d a t a n o t s h o w n ) .

Insulin bindiTuj to WGA purified receptors C r u d e r e t i n a l m e m b r a n e s w e r e s o l u b i l i z e d in 1 % T r i t o n X - 1 0 0 a n d p a r t i a l l y purified on WGA-agarose columns. Insulin binding to the solubilized but unpurified

828

R.J. W A L I ) I ~ I L L I G ET AL.

m e m b r a n e s w a s 8 " 0 - 1 0 " 0 % ; a n d t h e e I u t i o n profile for insulin b i n d i n g w a s t y p i c a l of insulin r e c e p t o r s f r o m o t h e r t i s s u e s (Fig. 2). [ ~ n I ] I n s u l i n b i n d i n g in t h e p e a k f r a c t i o n v a r i e d b e t w e e n 5 0 - 6 0 % a n d 2 - 3 % o f t h e insulin b i n d i n g w a s t'ecovered in t h e f l o w t h v o u g h . [ ~ a I ] I n s u l i n b i n d i n g in W G A - p u r i f i e d r e c e p t o r s (Fig. ~) w a s s i m i l a r to t h a t using (.*rude m e m b r a n e s . H a l f - m a x i m a l d i s p l a c e m e n t u s i n g u n l a b e l e d p o r c i n e insulin w a s 0'2 nM a n d w i t h h u m a n p r o i n s u l i n w a s 20 n ~ . L i v e r a n d brain m e m b r a n e s w e r e i n s o l u b l e in T r i t o n X-100 a n d t h u s b i n d i n g could n o t be m e a s u r e d in W G A purified r e c e p t o r s .

¢n

z .7.

60

z

20

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1000

10,000

FI('. 3. ~V(lA-l)urified insulin reeeptor.~ fi'om bovine retina: eompetition-inhilfition of specific [z"~IIinsulin binding, W(;A-purified insulin reeepto~ were incubated overnight (4°C) with [Z=~llinsulin (0"03 nSl) ill t h e absenue and presenee of variou.~ eolle,~ntratioml.~ o f unlal)eled p¢)reine insulin and human proinsulin. In the V,'IlA ti-aetion used |br this work. the specific binding (total binding minus nonspecific) in the ailsenee of unlabeled h~)rmone was 111"3%. The non-specilie I)in(ling (binding in the presence of a large excess, i.e. 1(1/tg ml-J ot' unlabeled insulin) was 18 % of the total binding, l('n0 is the concentration o f unhd)eled horm(me that produce.~ a half-maximal inhibition o f speeifie binding.

l'ho,~phor!thttion of the fl-.subunit and the tyro.sine-specific sub,strate Basal i n c o r p o r a t i o n o f 32|) i n t o t h e / t - s u b u n i t (90-95 k D a ) w a s d e m o n s t r a b l e ill ~,VGA-1)urified insulin r e c e p t o r s p r e p a r e d f r o m retinal m e m b r a n e s (Fig. 4). T h i s 3.2p i n c o r p o r a t i o n w a s f u r t h e r s t i m u l a t e d by insulin a t I0 -9- a n d I0-TM in a dosed e p e n d e n t f a s h i o n . A u t o r a d i o g r a p h s r e v e a l e d t h a t , in a d d i t i o n to a d e n s e b a n d o f r a d i o a c t i v i t y p r o d u c e d b y / ? - s u b u n i t . s w i t h a m o l e c u l a r w e i g h t in t h e v i c i n i t y o f 95 k D a , t h e r e w a s a g r a d i e n t o f d o w n w a r d l y d i m i n i s h i n g r a d i o a c t i v i t y (Fig. 4). T h e presence of this gradient may indicate a diminishing fi'equeney of small fl-subunits. T h e failure to e q u a l l y visualize t h e s m a l l e r / J ' - s u b u n i t m a y i n d i c a t e t h a t t h e s m a l l e r s u b u n i t s h a v e a s o m e w h a t lower s o l u b i l i t y in T r i t o n X - 1 0 0 or a lower ~,VGA affinity. T h e s m a l l e r , b u t n o t t h e l a r g e r , / / - s u b u n i t s were i m m u n o p r e e i p i t a t e d w i t h t h e antiinsulin r e c e p t o r a n t i b o d y B I 0 ( d a t a n o t s h o w n ) . T h i s s u g g e s t s t h a t t h e l a r g e r r e c e p t o r is n o n - c l a s s i c a l in t h e sense t h a t it h a s a u n i q u e a n t i g e n i c site. T h e f i n d i n g t h a t B 1 0 r e c o g n i z e s t h e s m a l l e r o f t h e b o v i n e insulin r e c e p t o r s , h o w e v e r , i n d i c a t e s t h a t species specificity of t h e a n t i b o d y is n o t a m a j o r c o m p l i c a t i n g f e a t u r e . F u r t h e r m o r e , u s i n g W G A - p u r i f i e d r e t i n a l i n s u l i n r e c e p t o r s , insulin s t i m u l a t e d t h e i n c o r p o r a t i o n o f a2p i n t o t h e e x o g e n o u s l y a d d e d artificial s u b s t r a t e p o l y (Gtu, T y r ) 4: 1. T h e i n s u l i n - s t i m u l a t a b l e p h o s p h o r y l a t i o n o f p o l y (Glu, T y r ) 4 : 1 w a s d o s e

I N S U L I N R E C E P T O R S IN B O V I N E R E T I N A

829

200-116-2--

Insulin (M)

0 10 - 9 10 - 7

Fro. 4. AutophosphoITlation of the fl-suburiit ot' the insulin receptor from the bovine retina. WGApurified receptors were preincubated for 30 rain at room temperature in the presence or absence of porcine insulin, a2P-labeled ATP mixture containing Mg2÷, Mn2÷, CTP and vanadate was added. After 10 rain the reaction was stopped and the samples were eleetrophoresed on SDS-polyaerylamide gels under reducing conditions. Autoradiography was used to localize the labeled subunit. d e p e n d e n t a n d was l i n e a r b e t w e e n 15 a n d 30 m i n (Fig. 5). H a l f - m a x i m a l s t i m u l a t i o n o c c u r r e d a t a n insulin c o n c e n t r a t i o n o f 2"6 nM.

Ftlctor 11111 activity T o d e t e r m i n e w h e t h e r r e t i n a l c r u d e m e m b r a n e p r e p a r a t i o n s were c o n t a m i n a t e d with blood vessels, t h e c e n t r i f u g a t i o n f r a c t i o n s o f w h o l e r e t i n a h o m o g e n a t e w e r e e x a m i n e d for f a c t o r V I I I i m m u n o f l u o r e s c e n c e . T h e pellets f r o m a n initial 600-, 2000-, 5000- or 1 0 0 0 0 g c e n t r i f u g a t i o n w e r e f o u n d to c o n t a i n n u m e r o u s i n t a c t blood vessels. In c o n t r a s t , m e m b r a n e s p r e p a r e d f r o m t h e s u p e r n a t a n t s o f t h e initial 2000-, 5000- a n d 10000 g centrifizgation w e r e f o u n d to be d e v o i d o f blood vessels. I n o n e e x p e r i m e n t , t h e s u p e r n a t a n t o f a 600-g c e n t r i f u g a t i o n h a d one blood vessel while t h e 600-£ pellet c o n t a i n e d 30 blood vessels. I n a second e x p e r i m e n t , t h e 600-g s u p e r n a t a n t h a d no blood vessels.

,~'ze of the insulin receptor ~z.subunit [12~I]Insulin was c r o s s l i n k e d t o r e c e p t o r s in c r u d e m e m b r a n e s p r e p a r e d f r o m whole n e u r a l r e t i n a . T h e [ ~ I ] i n s u l i n - i n s u l i n r e c e p t o r c o m p l e x w a s solubilized a n d r e d u c e d b y boiling t h e cross!inked m a t e r i a l (10 rain) in s a m p l e buffer c o n t a i n i n g 2 . 0 % S D S a n d 5"0% f l - m e r c a p t o e t h a n o l , F o l l o w i n g c e n t r i f u g a t i o n ( 1 2 0 0 0 g ; 1 5 r a i n ) , t h e s u p e r n a t a n t was a n a l y s e d u s i n g S D S - P A G E a n d a u t o r a d i o g r a p h y .

830

R.J. WALDBILLIG

E T AL.

1000 o

~ 4o_

-

/

oI'~

.~.. 4_.~_ ~

~ ,-.,,~,,,V" . . . . . . . . , .I

1.0

........ ~ ........ ~ ........

10 ng/ml

100

1000

10,000

F~o. 5. Time-course and dose-response of phosphorylation of artificial substrate poly (Glu, Tyr) 4 : 1 by WGA-purified insulin receptors from bovine retina. Receptors were i)re-incubated for 30 min at room temperature in the presence or absence of various concentrations of porcine insulin. The phosphorylation was initiated I)y adding Mg =+, CTP, A T P and ~[a=p]ATP. The phosphorylation reaction was measured at 15 and 30 rain. Data are expressed as insulin-induced incorporation of a=p (a=p incorporation into exogenous substrate in the presence of insulin minus the incorporation in the absence of insulin). a

b

c

d

e

f

g

h

0

0

i

j

k

200 --

Mr

(kDa) 116--

Insulin 10 -6 M

0

+

0

+

0

+

0

0

0

F1a. 6. Crosslinking of porcine [lZbl]insulin to the a sununit of insulin receptors from bovine retina. S D S - P A G E of [12aI]insulin crosslinked to insulin receptors in bovine tissue (except as noted [12hi|insulin was erosslinked to receptors in crude membrane and solubilized in SDS). Lanes a : liver-total binding ; b : liver-non-specific binding; c: brain-total binding; d : brain-non-specific binding; e: whole retina-total binding; f: whole retina-non-specific binding. Insulin receptors in whole retina crude membranes were erosslinked to [12hi|insulin and treated with Triton X-100; lane g shows the erosslinked receptors t h a t were insoluble in Triton X-100; lane i shows the crosslinked receptor t h a t was solubilized in Triton XI00 and then immunoprecipitated with the h u m a n polyclonal anti-insulin receptor a n t i b o d y BI0. Crude membrane from bovine whole retina were solubitized with Triton X-100 and WGA purified; h shows [125I]insulin crosslinked purified receptor. Lanes j and k show [l=sI]insulin crosslinked to retinal insulin receptors in crude membrane t h a t were (lane j) and were not (lane k) treated with neuraminidase.

INSULIN

RECEPTORS

I N F , ~ ) V I N E .~,'~, I N A

831

Radiographs revealed t h a t retinal insulin receptor :~l~ ~mit: !b'i,:. ~i. lane e ) f o r m a broad band of radioactivity (120-133 kDa) t h a t ov,,rI: !q)ed ~ , . ,ii~Iribution of a-subunits from brain (Fig. 6, lane c) and liver (Fig. 6, lane a) receptors. In some radiographs, a faint line of demarcation could be seen to divide the ¢¢-subunits into separate populations of larger ('liver-type ') and smaller (' brain-type ') receptors. The appearance of the ' liver-type' receptor in the distribution of subunits does not seem to be the result of vascular contamination. Factor V I I I d a t a revealed t h a t crude membranes prepared from the supernatant fractions of 2000-, 5000-, and 10000-g centrifugations are uniformly devoid of vascular tissue. Despite this, a-subunits from these and 600-g supermrtants show a pattern of heterogeneity, i.e. both liver- and brain-types of subunits (data n o t shown). The pattern of heterogeneity was i n d e p e n d e n t of w h e t h e r the membranes had been incubated with 3.0- or 0"03 FM [125I]insulin (data not shown). Also, it does not seem likely t h a t the appearance of the small ('brain-type') receptor is the result of overnight (4°C) proteo!ytie cleavage of the large# (' liver-type') receptor as the pattern of microheterogeneity in fresh tissue and t h a t stored overnight is not different (data not shown). In addition to having different molecular weights, the large and small insulin receptors are distinguishable by other criteria. For example, the larger ('liver') receptor appears to be more soluble in Triton X-100 than the small ("brain ') receptor. Figure 6 (lane g) shows t h a t the Triton X-i00-insoluble crosslinked material is mostly composed of smaller-sized receptors. The gradient of radioactivity t h a t extends toward the larger molecular weights m a y indicate t h a t the larger the receptor the more likely it is to be Triton X-100 soluble. Support for such a view is seen in Fig. 6 (lane h) where the Triton-solubilized (and WGA-purified) receptor is mostly the large (' liver-type ') receptor. The gradient of radioactivity (lane h) t h a t diminishes as it approaches the position of smaller receptors again associates the smaller receptor with diminished Triton X-100 solubility. I t is clear t h a t these differences in Triton X100 solubility are a m a t t e r of degree. Evidence t h a t some smaller receptors can be solubilized in Triton comes from the observation t h a t when the anti-insulin receptor antibody (B 10) is added to a preparation of solubilized receptors, the smaller receptor is immunoprecipitated (Fig. 6, lane e). The fact t h a t BI0 is less effective in ~

zoo

~

L

- v

kDo I16-•

Unlobelled insulin (nM)

0-0

:

0,I

....

.

0.5

:::

?::-,:

5.0

....

50

:

500

FIG. 7. Competition-inhibition of erosslinking of [x=bI]insulin to bovine retina. Crude membranes were incubated with 0"3 nM [x~sI]insulin overnight at 4°0 in the presence or absence of various concentrations of unlabeled porcine insulin. Following crosslinking with disuccinimidyl suberate, the crosslinked material was solubilized in sample buffer (2 % SDS) and run on polyacrylamide gels under reducing conditions. Following autoradiography, radiographs were analysed using scanning densitometry.

832

R.J. WALDBILLIG

ET AL.

immunoprecipita¢ing the large fbrm of the receptor (also present in the solubilized preparation) indicates another difference between these receptors. Finally, the two retinal insulin receptors appear to be different with respect to their glycosylation. Lanes j and k (Fig. 6) indicate t h a t the larger form of the insulin receptor is more sensitive to neuraminidase t r e a t m e n t than is the smaller. By conventional criteria, it appears t h a t both retinal receptors represent true insulin receptors since crosslinking with tracer levels (0"03 nM) of [12aI]insulin resulted in a pattern of microheterogeneity t h a t was not different from the pattern produced by incubation with 3"0 nM [12hi]insulin (data not shown). F u r t h e r support for this view is t h a t in crosslinking experiments, various concentrations of unlabeled insulin (0'1-500 nM) competed with [l~aI]insulin (0"3 nM) for binding with an IC~o in the nanomolar range (5 nM) (Fig. 7). Visually this competition appears equally effective in the two receptor subtypes. IGF-I was less p o t e n t than insulin in competing with [l~sI]insulin for binding (data not shown). 4. D i s c u s s i o n

H a v r a n k o v a , R o t h and Brownstein (2~978) first reported the presence of insulin binding sites in the rat retina. Since t h a t time. insulin binding sites have also been d e m o n s t r a t e d in embryonic chick retina (Peterson, Kyriakis and Hausman, 1986) and cultured cells from mouse retina (Thomopoulos and Pessac, 1979). Insulin receptors have also been partially characterized in cultured h u m a n retinoblastoma cells (Yorek, Speetor and Ginsberg, 1985; Savio|akis, Kyritsis and Chader, 1986). The vasculature from the neural retina t~as also been shown to contain insulin receptors (King, Goodman, Buzney, Moses and Kahn, 1985; Haskell, Meezan and Pillion, 1984). Im, Pillion and Meezan (1986) have recently reported on differential characteristics of asubunit binding in vascular and non-vascular retinal tissue. Yet, in spite of its potential physiological and clinical significance, a thorough characterization of the properties and characteristics of insulin receptors on neural cells of the retina has not been reported. We now show t h a t the bovine retina contains receptors meeting t h e conventional criteria for classification as insulin receptors. Specifically, the half-maxiinal inhibition of tracer binding in both crude m e m b r a n e s and WGA-purified receptors was in the low (0.1-1-0) nanomolar range. In crosslinking studies with crude membranes, insulin was more effective in inhibiting [125I]insulin (0"3 nM) binding than was IGF-I. As is typical of other insulin receptors (Hedo, Harrison and Roth, 1981), retinal insulin binding sites increased their affinity for insulin following solubilization and WGApurification. Furthermore, we have found t h a t these receptors are capable of undergoing insulin-induced autophosphorylation and possess a tyrosine-specific kinase activity (half-maximum 2-6 nM), characteristics typical of insulin receptors in other tissues (Zick, Kasuga, K a h n and Roth, 1983 ; Zick, Grunberger, Rees-Jones and Coral, 1985). Retinal insulin receptors are thus generally similar to those extensively studied in other tissues, notably liver and brain. Interestingly, however, retinal insulin receptors also exhit~it a n u m b e r of nonclassical characteristics. For example the a-subunit of ret{nal insulin receptors shows structural het¢ .' )geneity. More specifically, it appears t h a t there are two types of asubunits, one having a molecular size characteristic of bovine liver (133 kDa) and the other similar to bovine brain (120 kDa). Partial characterization of this heterogeneity

INSULIN RECEPTORS I N B O V I N E

RETINA

833

indicates t h a t retinal receptors exhibit differences in their glyeosylation. In this regard, neuraminidase t r e a t m e n t increased the mobility of the larger receptor in a m a n n e r typical of liver insulin receptors. On the other hand, the smaller retinal insulin receptor was relatively insensitive to neuraminidase, as is seen with brain insulin receptors (Hendricks, Agardh, Taylor and Roth, 1984; Heidenreich et al., 1986; Lowe and LeRoith, 1986; Shemer, Penhos and Lel¢oith, 19S6). This insensitivity to neuraminidase digestion suggests t h a t the smaller retinal insulin receptors have either fewer terminal sialic acid residues or, alternatively, t h a t these residues are inaccessible to neuraminidase. A n o t h e r non-classical feature of these insulin binding sites is t h a t the two receptors differ in their Triton X-100 solubility al~d recognition by the anti-insulin-receptor antibo(ly B 10. The smaller receptor is less soluble in Triton but appears to be preferentially recognized by BI0. In contrast, the large receptor' is Triton soluble but is not recognized by BI0. Although the large Triton-soluble receptor exhibits an insulin-stimulatable autophosphorylation (detectable at 1-0 riM) and an insulin-stimulatal)le phosphorylation of tyrosinecontaining substrate (half-maximum at 2"6 nM). the lack of recognition by 1310 indicates the need for further investigation of this non-traditional insulin binding site. It. is not clear" at tills point w h e t h e r the structural heterogeneity of these receptors indicates the presence of two receptors on a single cell type or, alternatively, represents a segregation of receptor by cell type (Lowe et al., I986). Insulin receptors of the liver type, for example, have been identified on bovine retinal microvessels (Haskell, Meezan and Pillion, 1984). I t seems unlikely, however, t h a t the heterogeneity observed here reflects contamination by vascular tissue, since crude m e m b r a n e preparations w i t h o u t blood vessels show clear microheterogeneity. Confirinatory evidence t h a t heterogeneity is not due to vascular contamination is t h a t a similar heterogeneity is seen in insulin receptors from highly |)urified bovine rod-outer segments (Waldbillig et al., 1987). A recent report (hn, Pillion and Meezan, 1986) failed to find microlleterogeneity in the neural retina insulin receptor. Their failure to d e m o n s t r a t e the large form of the insulin receptor m a y be due to: (1) centrifugation forces inadequate to pellet m e m b r a n e s containing the larger form of the receptor (their 2000g, 10 rain vs our 20000 g, 30 rain), (2) differential adsorption or elution of the two receptor types fi'om DEAE-ion exchange columns; or (3) differential solubility of the two receptors in Triton X-102. We have found t h a t in bovine tissue, the pattern of insulin receptor solubility is quite different f r o m t h a t found in other species (unpublished observations). For example, insulin receptors from bovine tJrain, liver, spleen and adrenal are insoluble in Triton X-100, CHAPS, nonidet, digitonin, octyl-glueoside and brij (Waldbillig and LeRoith, 1987). Even in the retina, where Triton X-100 is s o m e w h a t more effective, we have evidence t h a t the larger receptor is more soluble than the smaller. Therefore, the failure of hn, Pillion and Meezan (1986) to see the larger receptor may be related to its low solubility in Triton X-102. In this context then, our visualization of both forms of the retinal insulin receptor may be related to our vigorous solubilization procedure (boiling in 2'0% SDS). In s u m m a r y , insulin receptors are present on retinal neuronal cell membranes and exhibit m a n y of the structural and functional properties of insulin receptors from other tissues. However, the retina seems unique in t h a t it is a tissue with a heterogeneous population of insulin receptors.

834

R.J. W A L D B I L L I G ET AL. ACKNOWLEDGMENTS

We wish to thank Dr Jesse Roth for helpful suggestions in planning the experiments, and Violet Katz and Noreen Beavers for excellent secretarial assistance. REFERENCES Bradford, M. M. ~I976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye. Anal. Biochem. 72, 248-54. Ciaraldi, T., Robbins, R., Leidy, J. W., T h a m m , P. and Berhanu, P. (1985). Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors oil peripheral target cells. Endocrinology 116, 2179-85. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., On, J. H.~ Masia~z, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. and Rutter, W. J . (1985). The human insulin receptor cDNA : the structural basis for hormone-activated t r a n s m e m b r a n e signalling. Cell 40, 747-58. Haskell, J . F., Meezan, E. and Pillion, D. J. (1984). Identification and characterization of the insulin receptor of bovine retinal microvessels. Endocrinology 115, 696-704. H a v r a n k o v a , J., Roth, J. and Brownstein, M. (1978). Insulin receptors are widely distributed in the central nervous system of the rat. N a t u ~ (London) 272, 827-9. Hedo, J. A., Harrison, L. C. and Roth, J. (1981). Binding of insulin receptors to lectins: evidence for common carbohydrate determinants on sev~:al m e m b r a n e receptors. Biochemistry 20, 3385-93. Heidenreich, K . A . , Zahniser, N . R . , Berhanu, P., Brandenburg, D. and Ole£sky, J. M. (1983). Structural differences between insulin receptors in the brain and peripheral target tissues. J. Biol. Chem. 258, 8527-30. Hendricks, S, A., Agardh, C-D., Taylor, S. I. and Roth, J. (! 984). Unique features of insulin receptors in rat brain: J. Neurochem. 43, 1302-5. Im, J. H,, Pillion, D. J. and Meezan, E. (t986). Comparison of insulin receptors from bovine retinal blood vessels and non-vascular retinal tissue. Invest. Ophthalmol. Vis. Sci, 27, 1681-90. Kasuga, M., Akanuma, Y., Iwamoto, Y. and Kosaka, K. (1978). Insulin binding and glucose metabolism in adipocytes from streptozotocin-diabetic rats. A m . J. Physiol. 235, E 175-82. King, G. L., Goodman, A. D., Buzney, S., Moses, A. and Kahn, C. R. (1985). Receptors and growth-promoting effects o f insulin and insulin-like growth factors on cells from bovine retinal capillaries and aorta. J. Clin. Invest. 75, 1028-36. LeMarchand, Y., Loten, E. G., Assimacopoulos-Jeannet, F., Forgue, M. E., Freychet, P. and J e a n r e n a u d , B. (1976). Effect of fasting and streptozotocin on the obesehyperglycemic (ob/ob) mouse: Apparent lack of a direct relationship between insulin finding and insulin effects. Diabetes 26, 582-90. Lowe, W. L., Boyd, F. T., Clarke, D. W., Raizada, M. K., Hart, C. and LeRoith, D. (1986). Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and p r i m a r y cell cultures. Endocrinology 119, 25-35. Lowe, W. L. and LeRoith, D. (1986). Insulin receptors from guinea-pig liver and brain: structural and functional studies. Endocrinology 118, 1669-77. McElduff, A., Sehroer, J. A. and Taylor, S. I. (1984). Insulin bound to the insulin receptors of IM-9 lympocytes is more accessible to anti-insulin antibodies after t r e a t m e n t with dithiothreitol : bound insulin is deeply buried in the receptor-binding site. Endocrinology 115, 1869-71. Pacold, S. T. and Blackard, W. G. (1979). Central nervous system insulin receptors in normal or diabetic rats. Endocrinology 105, 1452-7. Peterson, S. x,V., Kyriakis, J . M. and H a u s m a n , R. E. (1986). Changes in insulin binding to developing embryonic chick neural retinal cells. J. Neurochem. 47, 851-5. Pilch, P. F. and Czech, M. P. (1980). The subunit structure of the high-affinity insulin receptors: evidence for a disulfide-linked receptor complex in fat cell and liver plasma membranes. J. Biol. Chem. 255, 1722-31.

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835

Puro, D. G. and Agardh, E. (1-984). Insulin-mediated regulation of neuronal maturation. Science 225, 1170-2. Rees-Jones, R. W., Hendricks, S. A., Quarum, M. and moth, J. (1984). The insulin receptors of rat brain is coupled to tyr.osine kincase activity. J. Biol. Chem. 259, 3470-4. Saviolakis, G. A., Kyritsis, A. P. and Chader, G. J. (1986). Human Y-79 retinoblastoma cells exhibit specific insulin receptors. J. Neurochem. 47, 70-6. Shemer, J., Penhos, J. and LeRoith, D. (1986). Insulin receptors in lizard brain and liver: structural and functional studies of a and fl subunits demonstrate evolutionary conservation. Diabetologia 29, 321-9. Simon, J. and LeRoith, D. (1986). Insulin receptors of chicken liver and brain: characterization of a and fl subunit properties. Eur. J. Biochem. 158, 125-32. Takayama, S., White, M. F., Lauris, V. and Kahn, C. R. (1984). Phorbol esters modulate insulin receptor phosphorvlation and insulin action in cultured hepatoma cells. Proc. Nat. Acad. Sci. U.S.A. 81, 7797-801. Thomopoulos, P. and Pessac, B. (1979). Insulin receptors in cultured mouse retina cells. Diabetologia 16, 275-9. Ullrich, A., Bell, J. R., Chen, E. Y., Hereto, R., Pet ruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.C.. Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld;C., Rosen, O. M. and Ramachandran, J. (1985). Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature (London) 313, 756-61. Waldbillig, R . J . , Fletcher, R.T., Chader, G.J., Rajagopalan, S., ~odrigues, M. and. LeRoith, D. (! 987). Retinal insulin receptors. II. Characterization and insulin-induced tyrosine kinase activity in bovine retinal rod outer segments. Exp. Eye Res. (in press). Waldbillig, R. J. and LeRoith, D. (1987). Insulin receptors in the peripheral nervous system: a structural and functional analysis. Brain Res..t4)9, 215-20. Yip, C. C., Moule, M. L. and Yeung, C. W. T. (1980). Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling. Biochem. Biophys, Re8. Commun. 96, 1671-5. Yorek, M. A., Spector, A. A. and Ginsberg, B. H. (1985). Characterization of an insulin receptor in human Y-79 retinoblastoma cells. Expt. Eye Res. 45, 1590-5. Zick, Y., Grunberger, G., Rees-Jones, R.W. and Coral, R. 5. (I985). Use of tyrosinecontaining polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine ldnases. Eur. J. Biochem. 148, 177-82. Zick, Y., Kasuga, M., Kahn, R. C. and Roth, J. (t983). Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell free system. J. Biol. Chem. 258, 75-80.