Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain

Developmental Brain Research, 42 (1988) 77-83

77

Elsevier BRD 50773

Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain Martine

Pomerance,

Jean-Michel Dani~le

Gavaret,

Toru-Delbauffe

Claude

Jacquemin,

Carole

Matricon,

and Michel Pierre

Unitd de Recherche sur la Glande Thyroi'de et la R~gulation Hormonale, 1NSERM U. 96, Kremlin-Bic~tre (France)

(Accepted 23 February 1988) Key words: Postnatal development; Insulin; Insulin-like growth factor 1; Receptor; Tyrosine kinase

The binding of insulin and insulin-like growth factor 1 (IGF1) to high-affinity sites in the brain of rats aged 2-37 days was studied. Specific binding of insulin and IGF1 was assessed using tracer concentrations of 125I-insulin or 125I-IGF1. Sites for insulin and IGF1 were distinguished in these conditions as shown by competition experiments. The Kd were 3.6 nM (insulin) and 2.0 nM (IGF1). These values did not change significantly over the age range studied. The numbers of high-affinity binding sites for insulin and IGF1 were similar in adult animals. IGF1 binding was higher than the insulin binding in 2-day-old animals. The binding capacity for both insulin and IGF1 decreased from birth to age 15 days and remained stable thereafter. Tyrosine kinase activity, which is associated with these receptors, was measured using the artificial substrate poly(Ghi, Tyr). It decreased over the first 15 days of life and remained stable thereafter. Autophosphorylation of the receptors confirmed this result. This decrease appears to be due to changes in the numbers of the two types of receptors, and is probably a reflection mainly of the variation in the number of IGF1 receptors. Similar results for insulin and IGF1 binding as well as tyrosine kinase activity were obtained with hypothyroid rats.

INTRODUCTION The presence of receptors for insulin and insulinlike growth factor 1 (IGF1) in the brain 6'12'17'22 suggests that these factors play a role in the central nervous system. But, despite much speculation, this role remains to be clearly demonstrated. The structure of insulin and IGF1 receptors consist of two a- and two fl-subunits linked by disulfide bonds. The mol. wt. of the a-subunit for the two receptors in brain is 115,000 Da 9'1°A4while the fl-insulin subunit h a s a mol. wt. of 94,000 Da 24 and the fl-IGF1 subunits have mol. wts. of 92,000 and 97,000 D a 15,21'26. The fl-subunits of both receptors display tyrosine kinase activity 23 suggesting that the molecular mechanism of action of the two factors is similar. The initial studies of the development of these receptors in the brain were those of Kappy et al. 16 on the insulin receptors. It now appears that specific insulin binding in rat brain increases at the end of the

gestation period and decreases in adult animals 19. It has also been reported that the intrinsic tyrosine kinase activity of the insulin receptor increased and then decreased at the same times 19. The binding of IGFs has been measured only in fetal human brain 22 and brain chick embryo 1. In the present work, we have examined the relative importance of insulin and IGF1 receptors in the rat brain during postnatal development. We have assayed insulin and IGF1 binding and the associated tyrosine kinase activities of the two receptors. The effect of thyroid status on the evolution of these receptors was also studied, since thyroid hormones play a fundamental role in brain development. MATERIALS AND METHODS Animals

Pregnant S p r a g u e - D a w l e y rats were maintained with a light:dark cycle of 12:12 h. On the 16th day of

Correspondence: M. Pomerance, Unit6 96 INSERM, 78, rue du G1. Leclerc, 94275 Le Kremlin-Bic~tre Cedex, France.

0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

78 gestation, half of the animals were fed a low-iodine diet. On the 18th day of gestation, 1 g/liter MTU (14hydroxy-6-methyl-2-thiopyrimidine) and 6 g/liter sucrose were added to the drinking water to block thyroid hormone synthesis. This treatment with MTU was maintained after birth until animals were sacrificed at 2-37 days of age by decapitation. The brains were quickly removed and the cerebral hemispheres were placed on ice.

Preparation of membranes Crude brain membranes were prepared as described by Havrankova et al. 12. These membranes (20,000 g pellet) were suspended in 50 mM HEPES pH 7.6, 10 mM MgC12. The membrane suspension was solubilized with 1% Triton X-100 (v/v) at 20 °C for 1 h in the presence of 1 mM PMSF (phenylmethylsulfonylfluoride), 100/~g/ml aprotinin, 1 keg/ml leupeptin and 10 mM 1-10 phenanthroline. Insoluble material was removed by centrifugation at 120,000 g for 1 h at 4 °C and the Clarified supernatant (1.5-4 mg/ml of protein) was stored at -70 °C. Wheat germ agglutinin chromatography Wheat germ agglutinin coupled to Ultrogel was purchased from IBF (France). Two ml of solubilized membranes were applied on 1 mi wheat germ agglutinin Ultrogel previously equilibrated with 50 mM HEPES, pH 7.6, 10 mM MgCI 2, 0.1% Triton X100 (v/v). The mixture was rotated end-over-end for 4 h at 4 °C. The flow-through was saved and the Ultrogel was washed first with equilibrating buffer and then with 100 ml of the same buffer containing 150 mM NaCI. Glycoproteins were eluted, after 1 h of end-overend rotation at 4 °C, with 0.3 M N-acetylglucosamine in 50 mM HEPES pH 7.6, 10 mM MgCI> 150 mM NaCI, 0.1% Triton-X100 (v/v), 1 mM PMSF, 100 #g/ml aprotinin, 1 /~g/ml leupeptin, 10 mM 1-10 phenantroline as previously described 13. Aliquots of the eluate, which contained insulin and IGF1 receptors, were stored a t - 7 0 °C in 10% glycerol (v/v). Proteins were determined by the Bradford method 4. Insulin or IGF1 binding assays t2sI-Insulin (230-290 Ci/mmol) was a generous gift of Dr. B. Desbuquois (H6pital Necker, Paris). Por-

cine insulin was obtained from Novo (Denmark). 125I-IGF1 (2000 Ci/mmol) and IGF1 were purchased from Amersham (England). The standard assay was performed in 100 mM HEPES pH 7.6, 120 mM NaCI, 1.2 mM MgSO4, 2.5 mM KC1, 15 mM sodium acetate, 1 mM EDTA. 0.1% BSA and 0.1% Triton. Aliquots of so|ubitized membranes (150-400 ktg/ml of protein) or wheatgerm agglutinin eluates (2-11/~g/ml of protein) were incubated at 4 °C for 16 h with a trace amount of 125Iinsulin or 125I-IGF1 (1.6 x 10-l° M) in a final volume of 0.2 ml. Non-specific binding was determined in the presence of excess unlabeled porcine insulin (2 x 10-s M). Bound and free 125I-ligand were separated by precipitation with polyethylene glycol (PEG, final concentration 12.5%) in the presence of y-globulin bovine (final concentration 0.05%) as described by Cautrecasas s. The mixture was left for 15 min at 4 °C, and precipitated proteins were filtered under reduced pressure on HVLP Millipore filters. The filters were washed with 8% PEG, and the retained radioactivity was measured in a Kontron gamma counter. Specific binding was calculated by subtracting non-specific binding from total binding.

Insulin or IGF1 receptor phosphorylation Samples of wheat germ agglutinin Ultrogel eluate were preincubated for 15 min at 24 °C in 50 mM HEPES pH 7.6, 150 mM NaCI, 10 mM MgCI 2, 2 mM MnCI2, 0.1% Triton X100 (v/v) and in the presence or absence of 10-7 M insulin or IGF1 (final volume of 30~1). Phosphorylation was initiated by adding 5 /~Ci [gP2p]ATP (3 Ci/mmol, Amersham, U.K.). After 10 min at 24 °C, the reaction was stopped by addition of concentrated electrophoresis sample buffer. The samples were heated at 100 °C for 4 min and analysed by SDS-PAGE according to Laemmli Is on a gel containing 12.5% acrylamide and 0.08% bis-acrylamide. After electrophoresis, the gels were fixed, dried a~d autoradiographed on Kodak X-Omat film. Molecular weights were determined by comparison with prestained molecular weight standards from Sigma. Artificial substrate phosphorylation The polymer (Glu4, T y r o used as the artificial sub-

79 strate was purchased from Sigma. Receptor samples were preincubated with or without insulin under conditions identical to those described for the autophosphorylation reaction. Exogenous substrate phosphorylation was initiated by the addition of 6/A of polymer (Glu4, Tyrl) at a final concentration of 2.4 mg/ml and 3/~Ci [y32p]ATP (3 Ci/mmol) in a final volume of 50/A and the mixture was incubated at 24 °C for 30 min. Phosphorylation was terminated by spotting 45 ~1 aliquots onto circles of W h a t m a n 3 MM filter paper, fixation in 10% trichloroacetic acid containing 3 m M A T P and extensive washing in 5% T C A . The filters were dehydrated in ethanol/ether 1/1 and ether, dried and counted by liquid scintillation. RESULTS

Comparison of insulin and IGF1 binding during postnatal brain development The insulin and IGF1 binding capacities of rat brains were assayed in animals aged 2 - 3 7 days. The results obtained with unsolubilized and Triton-X100solubilized m e m b r a n e proteins were similar for any given age or ligand. As there was complete solubilization of both receptors from young and adult animals, and as non-specific binding was greatly reduced in solubilized membranes, routine assays were performed on receptors solubilized with Triton X-100. U n d e r these assay conditions, insulin and IGF1 receptors were readily distinguished, as shown by the competition experiments (Fig. 1). Half-maximal inhibition of lZSI-insulin binding was achieved with 3 - 7 nM of unlabeled insulin whereas half-maximal inhibition of 125I-IGF1 was obtained with 1/~M insulin in both 2-day-old (Fig. 1A) and 37-day-old (Fig. 1B) animals. Similar results were obtained with hypothyroid rats (not shown). 10 nM of IGF1 also produced a 50% inhibition of 125I-IGF1 binding but no displacement of lzsI-insulin (not shown). The K d of insulin and IGF1 receptors for their respective ligands, determinated by Scatchard analysis, were 3.6 nM (insulin) and 2.0 nM (IGF1) in 21-day-old animals (Fig. 2). These K d values did not change significantly with animal age (2-37 days). The numbers of high-affinity binding sites (Bmax) were also determined by this method. In 21-day-old animals, the Bmax were 35 and 32 fmol/mg solubilized m e m b r a n e protein for IGF1

•. - 1 0 0

O O an

50 u. to

uJ

log INSULIN CONCENTRATION (M)

Fig. 1. Competition binding studies of IGF1 and insulin receptors from brain of 2-day-old (A) and 37-day-old (B) rats. 4.5 x 10TM M 125I-IGF1(©) or 3.5 x 10-1° M 125I-insulin (0) were incubated for 16 h at 4 °C with solubilized membrane extracts (28-80/~g of protein) in the presence or absence of various concentrations of unlabeled insulin (10-9 M to 10-6 M). Bound radioactivity was measured as described under Materials and Methods. The percentage of maximal specific binding of 125IIGF1 or 125I-insulinis plotted against the concentration of insulin unlabeled.

and insulin, respectively. The developmental patterns of insulin and IGF1 binding were examined using tracer concentrations of 125I-ligand. Fig. 3 shows a typical experiment. The binding of both ligands decreased rapidly between 2 and 15 days and remained constant thereafter. The binding of IGF1

i

i

I

T

I

i

~4.x == iu.v. m X~2r 5 ~00 BOUND

(pmol/I)

• Fig. 2. Scatchard plots of IGFI (O) and insulin (0) binding. Solubilized membrane receptors (from 21-day-old animals) were incubated with increasing concentrations of 125I-IGFI (6 x 10TM M to 9 x 10-1° M) or L25I-insulin(4 x 10-1° M to 3.2 x 10-9 M) in the presence or absence of excess unlabeled insulin (2 x 10-5 M) for 16 h at 4 °C. The ligand concentration range was appropriate for high-affinity binding sites only. Specifically bound radioactivity was determined as described under Materials and Methods and the bound/free ratio was calculated.

80

40,Z ,¢ .

o

©

= 8

Z

.3 D20I-

0

t

o~

1'0

2'0

3'0

4)0

AGE (days)

Fig. 3. Insulin (O) and IGF1 (O) binding as a function of rat age. 28-80/~g of solubilized membrane receptors (insulin and IGF1) were prepared from the cerebral hemispheres of 2- to 37-day-old rats. Solubilized proteins were incubated with a trace amount of 125I-insulinor 125I-IGF1(1.6 x 10-1° M) as described under Materials and Methods. Ligand specifically bound to receptors (fmol per mg of solubilized membrane protein) is plotted against age.

was higher than that of insulin in 2-day-old animals but the two bindings were similar in 37-day-old animals. The d e v e l o p m e n t a l p a t t e r n s for h y p o t h y r o i d rats were similar (not shown).

Evolution of the tyrosine kinase activity associated with insulin and IGF1 receptors Insulin- and I G F l - s t i m u l a t e d tyrosine kinase activity was m e a s u r e d with receptors which had been purified on w h e a t g e r m a g g l u t i n i n - U l t r o g e l as described under Materials and Methods. This chromatography p r o v i d e d the same recovery (70%) of solubilized insulin receptors as I G F 1 receptors and did not modify the I G F 1 receptor/insulin r e c e p t o r ratio. The tyrosine kinase activity was assayed using the artificial substrate poly(Glu4, Tyrl) in the presence of 10 7 M insulin, which stimulated the two receptors to a similar extent. Fig. 4 shows the variation in this activity (expressed p e r mg of m e m b r a n e - s o l u b i l i z e d proteins) as a function of age. A s with the binding of insulin and I G F 1 , it d e c r e a s e d b e t w e e n 2 and 15 days of age. Similar results were o b t a i n e d with hypothyroid rats. Fig. 5 shows the same p h e n o m e n o n visualized by a u t o p h o s p h o r y l a t i o n of the fl-subunits. A u t o phosphorylation of the eluates from agglutinin-U1trogel corresponding to the same a m o u n t of mere-

t'o

2'0

~0

AGE (days)

Fig. 4. Insulin- and IGFl-dependent tyrosine kinase activity assayed with the artificial substrate poly(Glu4, Tyrl) as function of age. Tyrosine kinase from the solubilized membranes of cerebral hemispheres from 2- to 37-day-old rats was purified on wheat germ agglutinin Ultrogel. Phosphorylation of poly(Glu4, Tyrl) was carried out as described in Materials and Methods. The difference between 32p incorporated into poly(Glu~, Tyrl) in the presence and absence of hormone, expressed as nmo132p incorporated per mg protein of solubilized membrane, is plotted against age.

brane proteins showed a lower activity in 15-day-old and 37-day-old animals than in n e w b o r n animals. Two p o l y p e p t i d e s were p h o s p h o r y l a t e d in the p r e s ence of insulin and this was particularly clear in the presence of I G F 1 (mol. wts. 92,000 and 97,000 D a ) . These bands p r o b a b l y c o r r e s p o n d to the two forms of fl-subunits previously o b s e r v e d in p r e p a r a t i o n s of IGF1 receptors 15,21. In these studies, we have not distinguished the tyrosine kinase activity associated with each of the two types of receptors. I n d e e d , as has been shown by Lowe and Leroith 2°, at h o r m o n e concentrations a b o v e 10 -9 M , the activities m e a s u r e d with each tigand were not additive and below 10 -9 M, we found a small and unprecise increase in tyrosine kinase activity p r o m o t e d by h o r m o n e . Nevertheless, in 2-day-old animals the tyrosine kinase activity stimulated with 10 -9 M IGF1 appears higher (50%) than that stimulated with 10 -9 M insulin. The activities stimulated by both ligands were similar in 37-day-old animals. Since fl-subunits of the two receptors are very similar 26, it seems reasonable to assume that the intrinsic tyrosine kinase activity of the two types of receptors is similar. In this case, the changes in tyrosine kinase

81

Fig. 5. Autophosphorylation of insulin and IGF1 receptor as function of age. Receptors purified on wheat germ agglutinin Ultrogel from the same amount of brain membranes of 2-, 15and 37-day-old rats were incubated without hormone (lanes 1, 4, 7), with 10 -7 M IGF1 (lanes 2, 5, 8) or with 10 -7 M insulin (lanes 3, 6, 9) for 15 min at 24 °C. Phosphorylation was performed for 10 min at 24 °C with [y-32p]ATPas described in Materials and Methods. Phosphorylated receptor preparations were analyzed by SDS PAGE and autoradiography.

activity associated with each type of receptors should parallel the variations in receptor number. Then, the decrease in total tyrosine kinase activity measured with poly (Glu 4, Tyrl) would be due mainly to IGF1 receptors. DISCUSSION These data indicate that, in the brains of newborn rats, there are more IGF1 receptors than insulin receptors and that the insulin and IGF1 binding capacities decrease during first 15 days of life. Lowe et al. 2° reported that insulin binding is lower in the brain of adult rats than in the brain of newborns. Their observations are in agreement with our results on insulin binding. Studies on the variation of IGF1 binding in the rat brain during postnatal development are missing. Our results indicate that only the number of binding sites changes. The affinity of neither receptor changes significantly. The Kd values of 3.6 nM for insulin receptor and 2.0 nM for IGF1 receptor are similar to those previously determined for rat brain and neuronal cells 5'9, but the ratio between insulin and

IGF1 receptors (1:1) in 37-day-old animals obtained in this study is different from that observed in adult rats (1:2.5) by Goodyer et al. n. This discrepancy may be due to differences in the ages of the animals or in the methods used to prepare membranes. The membranes used in our study were prepared in ammonium bicarbonate whereas sucrose was used in the study cited above. Tyrosine kinase activity changes in parallel with insulin and IGF1 binding. It decreases rapidly between days 2 and 15 of life, when measured by both autophosphorylation of the receptors and phosphorylation of the exogenous substrate poly(Glu 4, Tyrl). The tyrosine kinase activity assayed probably corresponds to the sum of the activities carried by the two types of receptor. As suggested above, the intrinsic activity of the two types of receptors might be similar. This hypothesis was also supported by other observations. Thus, in adult brain, in which we found a similar number of insulin and IGF1 receptors, 50% of the tyrosine kinase is lost during insulin receptor immunoprecipitation 2°. The 50% of remaining activity probably corresponds to IGF1 receptors. If insulin and IGF1 receptors have similar intrinsic activity, the changes observed would be mainly due to variations in the number of IGF1 receptors. Consequently, the suggestion of Lowe at al. 2° that there was a decrease in the intrinsic activity of the insulin receptor between birth and adulthood may require reconsideration since it might be the result of a simultaneous decrease in the number of IGF1 receptors, which had not been assayed. Hypothyroidy does not change the number, the affinity for their respective ligands, and the tyrosine kinase activities of the two receptors. However, it is always possible that thyroid hormones affect the insulin and IGF1 receptors on only a few cell types but cause no important change in the total number of these receptors in the whole brain. In this way, we have observed 7 that hypothyroidy increases the insulin-dependent tyrosine kinase activity of rat adipocytes. On another hand, endogenous production of these growth factors may be controlled by thyroid hormones, as suggested by the effect of T 3 on IGFs production by brain cells cultured in serum free-medium 2. The variations reported here in insulin and IGF1 binding and the associated changes in tyrosine kinase

82 activity have been o b s e r v e d with whole brain. It would clearly be interesting to examine the changes which take place in insulin and IGF1 receptors of neurons, astrocytes or o l i g o d e n d r o c y t e s in the various regions of the brain. T h e r e is already evidence that there are large differences between these cell types at the end of gestation. In fact, in cell cultures derived from the brains of n e w b o r n rats, insulin receptors are found in neurons 3, with almost none in glial cells (unpublished observations), whereas I G F 1 receptors are present in both neurons and glial cells 25. M o r e o v e r , changes in molecular weight of insulin and IGF1 a subunits during d e v e l o p m e n t have been also observed. These changes might be due to differences in glycosylation as suggested previously 25. With whole brain s'9'19 no clear difference in size has been r e p o r t e d b e t w e e n neonatal and adult brain but larger forms of receptors have been found in foetal brains. Burgess et ai. s have r e p o r t e d that an intermediate form of subunit of I G F 1 r e c e p t o r a p p e a r e d to be present in neonatal brain but they did not find this form in o n e - w e e k - o l d animals. H o w e v e r , in glial cells

REFERENCES 1 Bassas, L., De Pablo, F., Lesniak, M.A. and Roth, J., Ontogeny of receptors for insulin-like peptides in chick embryo tissues: early dominance of insulin like growth factor over insulin receptors in brain, Endocrinology, 117 (1985) 2321-2329. 2 Binoux, M., Falvre-Bauman, A., Lassarre, C., Barret, A. and Tixier-Vidal, A. Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium, Dev. Brain Res., 21 (1985) 319-321. 3 Boyd, F.T., Clarke, D.W., Mutuer, T.F. and Raizada, M.K., Insulin receptors and insulin modulation of norepinephrine uptake in neuronal cultures from rat brain, J. Biol. Chem., 260 (1985) 15880-15884. 4 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 5 Burgess, S.K., Jacobs, S., Cuatrecasas, P. and Sahyoun, N., Characterization of a neuronal subtype of insulin-like growth factor I receptor, J. Biol. Chem., 262 (1987) 1618-1622. 6 Chernausek, S.D., Jacobs, S. and Van Wyk, J.J., Structural similarities between human receptors for somatomedin C and insulin: analysis by affinity labeling, Biochemistry, 20 (1981) 7344-7350. 7 Correze, C., Pierre, M., Thibout, H. and Toru-Delbauffe, D., Autophosphorylation of the insulin receptor in rat adipocytes is modulated by thyroid hormone status, Biochem.

cultures derived from neonatal brain, receptors of intermediate size have been found 5'25. This difference might be due to culture conditions but it remains possible that some populations of cells in different regions of the brain have a particular subtype of receptors not easily detected in whole brain. The n u m b e r ef IGF1 receptors found in newborn rats suggest that this receptor type plays an important role in the brain at the end of gestation and just after birth. This idea is also s u p p o r t e d by reports that IGF1 binding is much higher than insulin binding in the h u m a n fetal brain 22 and in the brain of the chick e m b r y o ~. ACKNOWLEDGEMENTS W e are grateful to Dr. B. Desbuquois of the 1NS E R M U 30 (H6pital Necker, Paris) for his generous gift of t25I-insulin. W e also thank Mrs A. Lefevre and Mr M. Bahloul for the p r e p a r a t i o n of this manuscript. This work was s u p p o r t e d by grants from the Association pour la Recherche contre le Cancer and from the Universit6 Paris VII.

Biophys. Res. Commun., 126 (1985) 1061-1068. 8 Cuatrecasas, P., Isolation of the insulin receptor of liver and fat-cell membranes, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 318-322. 9 Gammeltoft, S., Haselbacher, G.K., Humbel, R.E., Fehlmann, M. and Van Obberghen, E., Two types of receptor for insulin-like growth factors in mammalian brain, Embo J., 4 (1985) 3407-3412. 10 Gammeltoft, S., Kowalski, A., Fehlman, M. and Van Obberghen, E., Insulin receptors in rat brain: insulin stimulates phosphorylation of its receptor subunit, FEBS Lett., 172 (1984) 87-90. 11 Goodyer, C.G., De Stephano, L., Lai, W.H., Guyda, H.J. and Posner, B.I., Characterization of insulin,like growth factor receptors in rat anterior pituitary, hypothalamus and brain, Endocrinology, 114 (1984) 1187-1195. 12 Havrankova, J., Roth, J. and Brownstein, M., Insulin receptors are widely distributed in the central nervous system of the rat, Nature (Lond.), 272 (1978) 827-829. 13 Hedo, J.A., Harrison, L.C. and Roth, J., Binding of insulin receptors to lectins: evidence for common carbohydrate determinants on several membrane receptors, Biochemistry, 20 (1981) 3385-3393. 14 Heidenreich, K.A., Zahniser, N.R., Berhanu, P., Bradenburg, D. and Olefsky, J.M., Structural differences between insulin receptors in the brain and peripheral target tissues, J. Biol. Chem., 258 (1983) 8527-8530. 15 Jacobs, S., Kull, F.C., Shetton Earp, Jr., H., Svoboda, M.E., van Wyk, J.J. and Cuatrecasas, P., Somatomedin C stimulates the phosphorylation of the fl-subunit of its own receptor, J. Biol. Chem., 258 (1983) 9581-9584.

83 16 Kappy, M., Sellinger, S. and Raizada, M., Insulin binding in four regions of the developing rat brain, J. Neurochem., 42 (1984) 198-203. 17 Kasuga, M., Van Obberghen, E., Nissley, S.P. and Rechler, M.M., Demonstration of two subtypes of insulinlike growth factor receptors by affinity cross linking, J. Biol. Chem., 256 (1981) 5305-5308. 18 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 19 Lowe, W.L., Boyd, F.T., Clarke, D.W., Raizada, M.K., Hart, C. and Leroith, D., Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures, Endocrinology, 119 (1986) 25-35. 20 Lowe Jr., W.L. and Leroith, D., Tyrosine kinase activity of brain insulin and IGF1 receptors, Biochem. Biophys. Res. Commun., 134 (1986) 532-538. 21 Morgan, D.O., Jarnagin, K. and Roth, R.A., Purification and characterization of the receptor for insulin-like growth

factor I, Biochemistry, 25 (1986) 5560-5564. 22 Sara, V.R., Hail, K., Misaki, M. and Fryklund, L., Ontogenesis of somatomedin and insulin receptors in the human fetus, J. Clin. Invest., 71 (1982) 1084-1094. 23 Sasaki, N., Rees-Jones, R.W., Zick, Y., Nissley, P.S. and Rechler, M.M., Characterization of insulin-like growth factor I stimulated tyrosine kinase activity associated with the fl-subunit of type I insulin-like growth factor receptors of rat liver cells, J. Biol. Chem., 260 (1984) 9793-9804. 24 Rees-Jones, R.W., Hendricks, S.A., Quarum, M. and Roth, J., The insulinreceptor of rat brain is coupled to tyrosine kinase activity, J. Biol. Chem., 259 (1984) 3470-3474. 25 Shemer, J., Raizada, M.K., Masters, B.A., Ota, A. and Leroith, D., Insulin-like growth factor I receptors in neuronal and glial cells, J. Biol. Chem., 262 (1987) 7693-7699. 26 Yu, K.T., Peters, M.A. and Czech, M.P., Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinase, J. Biol. Chem., 261 (1986) 11341-11349.