Chromaffin cells express two types of insulin-like growth factor receptors

Brain Research, 518 (1990) 95-100 Elsevier

95

BRES 15518

Chromaffin cells express two types of insulin-like growth factor receptors Anne Danielsen, Elisabeth Larsen and Steen Gammeltoft Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen (Denmark) (Accepted 7 November 1989) Key words: Adrenal medulla; Chromaffin cell; Insulin-like growth factor; Insulin-like growth factor receptor; Insulin-like growth factor-I receptor; Tyrosine kinase

The receptor binding, internalization and tyrosine kinase activation of insulin-like growth factors, IGF-I and IGF-II have been investigated in cultured adult bovine chromaffin cells. IGF-I receptor a-subunits (Mr -130,000) bound IGF-I and IGF-II with identical affinity (Kd - 1 nM) and insulin with about 1000 times lower affinity. IGF-II receptors (Mr -250,000) bound IGF-II with a Kd of 0.5 nM, IGF-I with about 10 times lower affinity and insulin with >10,000 times lower affinity. The amounts of IGF-I and IGF-II receptors on the cell surface were 8 × 104 and 4 x 104 sites per cell, respectively. Insulin bound to a specific receptor with K a - 2 nM and the amount of receptors was 1.5 x 104 sites per cell. IGF-I and IGF-II stimulated tyrosine kinase activity and autophosphorylation of the IGF-I receptor fl-subunit (Mr -94,000) with equal potency (EDso - 1 nM), whereas insulin was - 5 times less potent. Both IGF-I and IGF-II were internalized after their binding to cell surface receptors. Mannose-6-phosphate, which binds to the IGF-II receptor, did not alter the binding or internalization of IGF-II. It is concluded that IGF-I and IGF-II can exert their biological effects in chromaffin cells by activation of the IGF-I receptor tyrosine kinase or by interaction with the IGF-II receptor. INTRODUCTION Insulin-like growth factors IGF-I and I G F - I I are polypeptides which show a structural similarity with insulin and act as mitogens on various cell types 14'21. In the developing rat and human embryo, IGF-I and I G F - I I are synthesized in a variety of tissues, suggesting that I G F ' s are involved in the regulation of fetal growth 2,17 During postnatal life, I G F - I is mainly secreted by the liver to the circulation under the control of growth h o r m o n e and stimulates skeletal growth 9, whereas I G F - I I is secreted by the choroid plexus to the cerebrospinal fluid and may exert neurotrophic functions via IGFreceptors in the brain 11A9'22'35. Recently, gene and protein expression of I G F - I and I G F - I I have also been shown in other tissues of adult man and rat 3'15'18'26. A m o n g these tissues is the adrenal gland where I G F - I I m R N A and immunoreactive I G F - I I has been isolated from the human adrenal medulla and pheochromocytoma, whereas IGF-I could not be detected 2°. This suggests that IGF-II may be involved in paracrine regulation of chromaffin cell activity in adult mammals. Thus, IGF-II may be characterized as a neuroactive factor in the central and peripheral nervous system of adult mammals 13. A prerequisite for the actions of IGFs in chromaffin cells is the presence of specific receptors on the cell surface. Two types of I G F receptors are present on many cell types

including cells in the central nervous system 11A3'3°. IGF-I receptors are heterotetramers composed of two a-subunits (M r -130,000) and two fl-subunits (M r -95,000) which show tyrosine kinase activity, whereas IGF-II receptors are monomers (Mr -250,000) which are identical with the mannose-6-phosphate receptor 14'33. The mannose-6-phosphate receptor has been implicated in intracellular transport of phosphomannosylated lysosomal enzymes from the Golgi apparatus and plasma membrane to the lysosome 39. The IGF-II receptor mediates endocytosis of IGF-II, whereas the IGF-I receptor seems to be involved in intracellular signalling of IGF-I and IGF-II 14. Recently, we have described that rat PC12 pheochromocytoma cells express two types of I G F receptors and that IGF-I and IGF-II are potent mitogens in this cell line 27. Others have identified a specific IGF-I receptor on adult bovine chromaffin cells which mediates a stimulatory effect of IGF-I on potassium-evoked catecholamine secretion 6. The fact that IGF-II, but not IGF-I, is produced by the adult human adrenal medulla led us to investigate the interaction of IGF-II with both types of I G F receptors in cultured bovine chromaffin cells. MATERIALS AND METHODS Materials Recombinant human IGF-I and IGF-II~° were a generous gift from M.C. Smith, Lilly Research Labs., Indianapolis, U.S.A.

Correpondence: S. Gammeltoft, Department of Clinical Chemistry, Bispebjerg Hospital, DK 2400 Copenhagen NV, Denmark. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

96 [12"Sl]IGF-I and [J2sI]IGF-II were iodinated by the chloramine T method at a specific activity of 40-60 ,uCi//~g4°. Human insulin and [A14-125I]monoiodoinsulin (250 ktCi//~g) were gifts from NOVO Research Institute, Copenhagen, Denmark. [7 -32p]ATP was purchased from Amersham, U.K. Disuccinimidyl suberate was from Pierce, Rockford, IL, U.S.A. Wheat germ agglutinin sepharose was from Pharmacia, Uppsala, Sweden. Poly-Glu-Tyr (1:4) was from Sigma, St. Louis, U.S.A. Monoclonal antibody to tyrosine hydroxylase was purchased from Boehringer Mannheim, F.R.G. Antiserum to fibronectin was purchased from Dakopatts, Copenhagen, Denmark. Dulbecco's modified Eagle's medium, Hams F-12 medium, and fetal calf serum (heat-inactivated) were purchased from Gibco, U.K. Multidishes and flasks for cell culture were obtained from NUNC, Roskilde, Denmark,

purified by affinity chromatography on wheat germ agglutinm sepharose. Tyrosine kinase activity was measured by incubation of glycoproteins at 20 °C with various concentrations of IGF-I, IGF-II or insulin for 90 min, followed by addition of 10 mg/ml poly-Glu-Tyr for 10 min and 4 mM MnCI2, 8 mM Mg C12, 20/~M ATP and 8 #Ci/ml [7 -32p]ATP for 30 min. The phosphorylation reaction was stopped by precipitation of proteins on filter paper in 10% trichloroacetic acid and 10 mg/ml sodiumpyrophosphate and the incorporated 32p-actitivy was counted in a fl-counter. Autophosphorylation of IGF-I receptors was studied by omitting the substrate, poly-Glu-Tyr from the reaction mixture and analyzing the 32p-labelled proteins by SDS-PAGE under reducing conditions followed by autoradiography 1

Internalization assay Cell culture Bovine chromaffin cells were purified and cultured as described by Livett et al. 24. In brief, bovine adrenal glands were digested with collagenase and the chromaffin cells were separated from the adrenocortical cells and non-viable cells on a Percoll density gradient. Further purification of chromaffin cells was achieved by differential plating of the cells for 2 h, whereby adhering fibroblasts were separated from non-adhering chromaffin cells37. The chromaffin cells were plated at a density of 1-2 × 105 cells per cm2 on poly-o-lysine-coated dishes in a 1:1 mixture of Dulbecco's modified Eagle's medium and Hams F-12 medium buffered with 15 mM HEPES and 11 mM NaHCO 3 supplemented with 10% fetal calf serum. In some chromaffin cell cultures 2.5/~g/ml fluorodeoxyuridine and 2.5/~g/ml cytosine arabinoside were included to inhibit cell proliferation. After 2 days in culture, the medium was removed and the culture continued for 2 days in medium without serum. The content of chromaffin cells and fibroblasts in the culture was determined at the time when the cells were used in binding experiments. More than 90% of the cells were stained with antibody to tyrosine hydroxylase whereas less than 10% were stained with antibody to fibronectin using fluorescence microscopy. Primary cultures of fibrobtasts from bovine adrenal medulla were prepared by differential plating and cultured in the same medium for 2-4 weeks. Cell protein was determined by the BioRad assay 5.

Binding assay Receptor binding of [125I]IGF-I, [a25I]IGF-II and [125I]insulin was measured on cell monolayers in a poly-D-lysine-coated 24-well multidish as described elsewhere 12. The cells were incubated at 4 °C with 150 pM of [125]IGF-I or [125I]IGF-II, or 25 pM of [125I]insulin ( - 105 cpm/ml) and various concentrations of unlabelled peptides in 250 #1 Krebs-Ringer buffer with 50 mM HEPES (pH 7.4) and 10 mg/ml bovine serum albumin. After 5 h, steady state was obtained and the cells were washed 3 times with ice-cold buffer, solubilized in 0.2 M NaOH and the radioactivity was counted in a 7-counter.

Affinity labelling of receptors For affinity labelling cell monolayers in 25 cm 2 flasks were incubated at 4 °C for 5 h with 3 nM [125I]IGF-I or [12sI]IGF-II ( - 2 × 106 cpm/ml) in the absence or presence of 0.1 /~M unlabelled IGF-I or IGF-II, respectively. The cells were washed twice with ice-cold buffer without albumin and peptides followed by crosslinking with 0.1 mM disuccinimidyl suberate for 15 min at 4 °C. After quenching of the reaction with 10 mM Tris buffer (pH 7.4), the labelled proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDS-PAGE) under reducing conditions and autoradiography for two months 12.

Tyrosine kinase assay Protein tyrosine kinase activity was measured as previously described ~. Chromaffin cells cultured in five 175 cm 2 flasks were scraped off and solubilized in 150 mM NaC1, 50 mM HEPES (pH 7.6) and 1% w/v Triton X-100 with addition of 0.17 mg/ml phenyl-methyl-sulfonyl-fluoride, 1.8 mg/ml bacitracin, and 100 KIE/ml aprotinin by gentle stirring for 60 min at 4 °C. After ultracentrifugation at 105 g for 90 min, the glycoproteins were

Internalization of receptor-bound IGF-I or IGF-I1 was analyzed by the acid-wash technique TM. After binding of 0.3 nM 125I-labelled IGF-I or IGF-II at 37 °C, surface-bound radioactivity was released by incubation with 0.2 M acetic acid and 0.5 M NaCI (pH 3.5) followed by solubilization of internalized radioactivity in 0.2 M NaOH. RESULTS

Receptor binding of IGF-I, IGF-H and insulin C o m p e t i t i v e binding e x p e r i m e n t s at 4 °C s h o w e d that c h r o m a f f i n cells h a v e two types o f I G F r e c e p t o r s as well as insulin r e c e p t o r s on t h e i r cell surface (Fig. 1). B i n d i n g of [125I]IGF-I was i n h i b i t e d by I G F - I and I G F - I I with e q u a l affinity ( K d - 1 n M ) , w h e r e a s insulin was a b o u t 1000 times less p o t e n t . B o u n d [125I]IGF-II was i n h i b i t e d by I G F - I I with a K d of - 0 . 5 n M , I G F - I b e i n g a p p r o x i m a t e l y 10 t i m e s less p o t e n t and insulin > 1 0 , 0 0 0 t i m e s less p o t e n t . Finally, binding of [125]insulin was i n h i b i t e d by insulin and I G F - I I with the s a m e affinity (Kd --2 n M ) w h e r e a s 1GF-I s h o w e d no inhibition at 10 n M suggesting a lower

affinity.

The

number

of

receptors

c h r o m a f f i n cell surface was d e t e r m i n e d

on

the

by S c a t c h a r d

analysis of the binding d a t a (Fig. 2). T h e t h r e e p e p t i d e s b o u n d to single classes of r e c e p t o r s w h i c h w e r e p r e s e n t in n u m b e r s of 8 x 104sites/cell ( I G F - I ) , 4 x 104 sites/cell ( I G F - I I ) and 1.5 × 104 sites/cell (insulin). T h e binding characteristics of I G F - I , I G F - I I and insulin r e c e p t o r s w e r e identical in c h r o m a f f i n cell cultures g r o w n in the absence or p r e s e n c e of f l u o r o d e o x y u r i d i n e and cytosine arabinoside

(data

not

shown).

For

comparison,

the

r e c e p t o r binding was studied w i t h 100% p u r e fibroblasts p r e p a r e d f r o m the b o v i n e a d r e n a l m e d u l l a . T h e I G F - I r e c e p t o r b o u n d I G F - I and I G F - I I with e q u a l affinity (Kd 1 n M ) and was p r e s e n t in a n u m b e r of 5 x 104 sites/cell. The IGF-II receptor bound IGF-II with K d -3 nM w h e r e a s I G F - I c r o s s - r e a c t e d with > 10,000 l o w e r affinity. T h e n u m b e r of I G F - I I b i n d i n g sites on fibroblasts was a b o u t 5 x 105 sites/cell. T h u s , the I G F - I r e c e p t o r was similar in the c h r o m a f f i n cell and f i b r o b l a s t cultures, w h e r e a s the I G F - I I r e c e p t o r s h o w e d d i f f e r e n c e s ( d a t a not shown). B a s e d on t h e s e findings we c o n c l u d e that the r e c e p t o r b i n d i n g s t u d i e d with t h e c h r o m a f f i n cell culture r e p r e s e n t s I G F - I and I G F - I I r e c e p t o r s on the c h r o m a f f i n

97

SPECIFICITY OF I G F - I . I G F - I I AND INSULIN RECEPTORS ON BOVINE CHROMAFFIN CELLS AT 4"C 125

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Fig. 1. Specificity of IGF-I, IGF-II and insulin receptors on cultured bovine chromaffin cells. Cell monolayers in 24-well dishes (2 x 105 cells per well) were incubated with 150'pM (-105 cpm/ml) of 125f-labelled IGF-I, IGF-II or insulin in the presence of various concentrations of unlabelled IGF-I (O), IGF-II (O) or insulin (A). Bound radioactivity was determined after 5 h at 4 °C. The receptor binding was calculated by subtraction of the non-specific binding determined in the presence of 0.1 /~M IGF-I or IGF-II, or 1 /~M insulin, respectively. The non-specifically bound tracer was about 5-10% of total binding. The results are mean of 4 experiments with S.E.M. indicated as bars. cells and not on the < 1 0 % contaminating fibroblasts.

Affinity labelling of IGF-1 and IGF-H receptors Affinity labelling of I G F receptors on the surface of chromaffin cells showed specific labelling of two proteins of M r - 1 3 0 , 0 0 0 and M r ~250,000 after reduction (Fig. 3). [125I]IGF-I labelled preferentially the 130,000 protein, whereas [125I]IGF-II labelled both proteins, the 250,000 stronger than the 130,000 protein. Taken together with the specificity of the competitive binding experiments, our d a t a suggest that chromaffin cells express I G F - I and I G F - I I receptors as well as insulin receptors.

studied in a p r e p a r a t i o n of detergent-solubilized and wheat germ agglutinin-purified glycoproteins from chromarlin cells by p h o s p h o r y l a t i o n of the synthetic substrate poly-Glu-Tyr. A s shown in Fig. 4, a tyrosine kinase was stimulated 2-fold by I G F - I and I G F - I I with almost similar potency (EDs0 - 1 nM), whereas insulin stimulated tyrosine p h o s p h o r y l a t i o n with slightly lower potency. In addition, I G F - I and I G F - I I stimulated the phosphoryla-

AFFINITY LABELLING OF IGF RECEPTORS IN BOVINE C H R O M A F F I N CELLS OR

IGF-I receptor kinase activity The tyrosine kinase activity of the I G F - I r e c e p t o r was

SCATCHARDANALYSISOF IGF-I.IGF-II ANDINSULIN RECEPTORSON BOVINE CHROMAFFINCELLS ~0.5.

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100 200 300 400 500 BOUND(pmol/g cell protein) Fig. 2. Scatchard analysis of IGF-I, IGF-II and insulin receptors. Receptor-bound IGF-I (O), IGF-II (O) or insulin (&) shown in Fig. 1 were analyzed according to Scatchard34, and the Kd values calculated from the slopes. The intercepts on the abscissa equal the amounts of receptors. Data are mean of 4 experiments with S.E.M. indicated as bars.

Fig. 3. Affinity labelling of IGF-I and IGF-II receptors in cultured chromaffin cells. Cell monolayers in 25 cm2 flasks (5 x 106 cells) were incubated with 3 nM ( - 2 x 106 cpm/ml)~25I-labelled IGF-I (left panel) or IGF-II (right panel) in the absence or presence of 0.1 aM unlabelled IGF-I or IGF-II, respectively. After 5 h at 4 °C the receptor-bound 125I-labelled peptide was cross-linked with 0.1 mM disuccinimidyl suberate for 15 min at 4 °C. The cells were scraped off, boiled 5 min in 3% SDS and 100 mM dithiothreitol, and analyzed by SDS-PAGE followed by autoradiography. OR, origin of resolving gel.

98

TYROSINE KINASE ACTIVITY OF PARTIALLY

INTERNALIZATIONOF IGF-I AND IGF-II IN BOVINE CHROMAFFINCELLS

G PURIFIED RECEPTORS FROM CHROMAFFIN CELLS .J

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Fig. 4. Tyrosine kinase activity of solubilized IGF-I and insulin receptors from chromaffin cells. Wheat germ agglutinin-purified glycoproteins were incubated with various concentrations of IGF-I (O), IGF-II (C)) or insulin (&) for 2 h at 20 °C followed by addition of 10 mg/ml poly-Glu-Tyr for 10 min, 4 mM MnC12, 8 mM MgCI e, 20 ~M ATP and 8 /~Ci [7 -32P]ATP for additional 15 min. The phosphorylation reaction was terminated by precipitation of 32p_ labelled proteins on filter paper with 10% trichloroacetic acid. The incorporated radioactivity was counted in a fl-counter. The data are representative of 3 experiments with similar dose-response curves but variable maximal response between 50 and 100% over basal activity.

tion of a protein (M r -94,000) approximately 5-fold as demonstrated by S D S - P A G E and autoradiography (Fig. 5). Insulin was significantly less potent. These data suggest that IGF-I and IGF-II activates the IGF-I receptor kinase which phosphorylates its own 3-subunit

AUTOPHOSPHORYLATION OF IGF'I RECEPTOR FROM BOVINE CHROMAFFIN CELLS OR

--

200

--

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66

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Fig. 5. Autophosphorylation of IGF-I and insulin receptors. Wheat germ agglutinin-purified glycoproteins were incubated with buffer, IGF-I, IGF-II or insulin in a concentration of 10 nM for 2 h at 20 °C followed by addition of 4 mM MnCI2, 8 mM MgCI 2, 20pM ATP and 8 pCi [X JzP]ATP for 15 min. The samples were boiled 5 min in 3% SDS and 100 mM dithiothreitol and analyzed by PAGE and autoradiography. OR, origin of resolving gel.

Q

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SURFACE BOUND

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Fig. 6. Internalization of IGF-I and IGF-II by chromaffin cells. Cell monolayers in 24-well dishes (4 × 105 cells per well) were incubated with 0.3 nM [125I]IGF-I (upper panel) or [12sI]IGF-II (lower panel) for various times at 37 °C. Surface-bound radioactivity ( 0 ) was measured by acid-washing the cells with 0.2 M acetic acid and 0.5 M NaCI and internalized radioactivity (C)) by solubilization of the cells with 0.2 M NaOH. The data were corrected for non-specifically bound or internalized radioactivity determined in parallel incubations with of 0.1 #M unlabelled IGF-I or IGF-II. Data are representative of 3 experiments with similar time courses, but variable degree of internalization between 50 and 80% of total binding.

and an exogenous substrate on tyrosine residues, whereas insulin activates the insulin receptor kinase.

Internalization of IGF-I and IGF-H The internalization of surface-bound [lZSI]IGF-I was studied by the acid-wash technique. Fig. 6 shows that surface-bound (acid-sensitive) IGF-I increased rapidly at 37 °C to reach a constant level after - 2 0 min. This was followed by a slower rise in internalized (-acid-resistant) radioactivity which reached a constant level after - 4 0 min. At steady state approximately 20% of total cell-associated 1GF-I was inside the cell. Surface-bound [125I]IGF-II was intemalized to the same extent as [12sI]IGF-I although at a slower rate as seen in Fig. 6. The amounts of internalized IGF-I and IGF-II varied between 50 and 80% of total cell-associated radioactivity in different preparations of chromaffin cells. Addition of 5 mM mannose-6-phosphate did not increase the binding or internalization of IGF-II, as seen previously in fetal rat brain neurons 28, suggesting that the two ligands do not interact on their common receptor in chromaffin cells (data not shown).

99 DISCUSSION The IGF-II gene and protein is expressed in the adrenal medulla and pheochromocytoma of adult humans 2°, and cultured adult bovine chromaffin cells express IGF-II m R N A (Danielsen, Larsen, Nielsen and Gammeltoft, unpublished). In the human fetus, both IGF-I and IGF-II transcripts are present in the connective tissue of the adrenal gland as demonstrated by in situ hybridization 17. These observations suggest that IGF-I and IGF-II may exert their actions in the developing and mature adrenal medulla in a paracrine manner. Alternatively, IGFs may be secreted to the circulation and act in other tissues. A prerequisite for the biological actions of IGF-I and IGF-II in chromaffin cells is the presence of specific I G F receptors on the cell surface. In the present study, we have demonstrated that two types of I G F receptors are present on cultured adult bovine chromaffin cells. IGF-I and IGF-II bind to the IGF-I receptor and activates its tyrosine kinase with equal potency. In addition, we have identified an IGF-II receptor which binds IGF-II with high affinity. Both IGF-I and IGF-II are internalized following their binding to receptors on the cell surface. Our findings indicate that IGF-II synthesized in the adult adrenal medulla of man and cow may act on chromaffin cells via interaction with two types of IGF receptors. Studies of the cellular mechanism of action of IGF-I and IGF-II in various cell-types have demonstrated that the IGF-I receptor tyrosine kinase is involved in the metabolic and growth-promoting effects of both peptides 1'8, whereas a regulatory function of the IGF-II receptor has been unclear 33. Our finding that IGF-II binds to the IGF-I receptor in chromaffin cells and activates the tyrosine kinase with the same potency as IGF-I, suggests that the cellular actions of IGF-II are mediated by the IGF-I receptor. The role of the IGF-II receptor could be to remove extracellular IGF-II by endocytosis and proteolytic degradation as described in fetal rat brain neurons ~. At present the nature of the biological effects of IGF-II in the adult mammalian adrenal medulla is not clear. Recently, it has been reported that IGF-I interacts with a specific IGF-I receptor and enhances the potassiumevoked catecholamine secretion from cultured bovine chromaffin cells 6. In other cell types of neuronal origin, various stimulatory actions of IGFs and insulin have been reported, including norepinephrine uptake in fetal rat brain neurons 4, acetylcholine release from adult rat brain cortical slices 29, and neurite outgrowth and cell proliferREFERENCES 1 Ballotti, R., Nielsen, EC., N., Kowalski, A., Richardson, W.D., Van Obberghen, E. and Gammeltoft, S., Insulin-like growth factor I in cultured rat astrocytes: expression of the gene and

ation in human neuroblastoma cell lines 25'32. Furthermore, we and others have demonstrated that rat PC12 pheochromocytoma cells express two types of I G F receptors and that both IGF-I and IGF-II stimulate proliferation of PC12 cells with high potency 7'27. In the present study, however, adult bovine chromaffin cells did not proliferate under basal or IGF-stimulated conditions as measured by double immunocytochemical staining of incorporated bromodeoxyuridine and tyrosine hydroxylase. It seems likely that chromaffin cells are postmitotic in the adult adrenal medulla. In contrast, IGF-I and IGF-II bound to specific receptors and stimulated the division of cultured bovine fibroblasts which were separated from the chromaffin cells by differential plating and identified with an antibody to fibronectin (Danielsen, Larsen and Gammeltoft, unpublished). It should be added, that IGF-I receptors have been identified on cultured bovine adrenal fasciculata cells and that IGF-I enhances the steroidogenic response to A C T H and angiotensin-I131. Thus, IGFs may exert multiple functions in the adrenal gland. Other growth factors like nerve growth factor, ciliary neurotrophic factor, basic fibroblast growth factor and epidermal growth factor have been shown to interact with cultured chromaffin cells from the young postnatal rat, and to induce effects like increased survival, cell proliferation, neurite outgrowth, induction of tyrosine hydroxylase and increased catecholamine content 23'36'38. Thus, the function of chromaffin cells seems to be regulated during development and postnatal life by several growth factors including IGF-I and IGF-II. In conclusion, our demonstration that IGF-I and IGF-II bind to two distinct types of I G F receptors on cultured bovine chromaffin cells suggests that IGFs are potentially important modulators of chromaffin cell function and that IGF-II synthesized in the adrenal medulla acts by a paracrine mechanism on chromaffin cells. Acknowledgements. Lotte Vogel, Finn Cilius Nielsen and Phil Marley are thanked for valuable help and discussions during the study. Michele C. Smith is gratefully acknowledged for the gift of recombinant IGF-I and IGF-II. Birte Kofoed is thanked for technical assistance, Merete Jacobsen for secretarial assistance and Lisbeth Jensen for the drawings. This study was supported by Danish Medical Research Grants 12-7725 and 12-6062 and by grants from the Danish Cancer Society, The NOVO Foundation, the Danish Hospital Foundation, and the Foundation for Advancement of Medical Research. S.G. was supported by The Danish Biotechnology Center for Neuropeptide Research. E.L. and A.D. both received research scholarships from the Medical Faculty, University of Copenhagen, and have contributed equally to the present study. receptor tyrosine kinase, EMBO J., 6 (1987) 3633-3639. 2 Beck, E, Samani, N.J., Penschow, J.D., Thorley, B., Tregear, G.W. and Coghlan, J.P., Histochemical localization of IGF-I and -II mRNA in the developing rat embryo, Development, 101 (1987) 175-184.

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