DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 7(2):207-216, 1990
CHARACTERIZATION OF TYPE II INSULIN-LIKE GROWTH FACTOR (IGF) RECEPTORS IN SHEEP LIVER PLASMA MEMBRANES K. L. Hossner and R. S. Yemm Department of Animal Sciences Colorado State University Fort Collins, Colorado 80523, U.S.A. Received July 21, 1989 ABSTRACT Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of tz~I-hlGF-lI (40%) to liver plasma membranes greatly exceeded that of t251hIGF-I (1.5%) after incubation at 20 C for 90 rain. Binding of Iz~I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of t2SI-hlGF-II was only partially reversed by addition of 1 O0 nM IGF.II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of tzSI-hlGF-II binding demonstrated that IGF.II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 12SI-hIGF-II. Plasma membranes were affinity cross-linked to :25I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,O00-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band..These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type lI receptors. INTRODUCTION Insulin-like g r o w t h factors (IGFs) are g r o w t h - p r o m o t i n g p o l y p e p t i d e s w h i c h are c h e m i c a l l y and b i o l o g i c a l l y related to insulin. Circulating serum IGF's are t h o u g h t to b e p r o d u c e d primarily by the liver (1, 2), although several tissues have b e e n s h o w n to synthesize these p o l y p e p t i d e s (3, 4). G e n e r a l l y c o n s i d e r e d e n d o c r i n e factors, the IGF's also act via a u t o c r i n e a n d / o r paracrine mechanisms (5,6) and r e c e p t o r s for these growth factors are present in many tissues, i n c l u d i n g the liver ('7, 8). Insulin-like g r o w t h factors have b e e n s h o w n to stimulate the i n c o r p o r a t i o n o f glucose into liver cell glycogen, e n h a n c e RNA and p r o t e i n synthesis (9) and e n h a n c e s e c r e t i o n o f a l b u m i n and h e m o p e x i n in c h i c k e m b r y o hepatocytes ( 1 0 ) . Similar effects o n g l y c o g e n synthesis have b e e n s h o w n in h u m a n h e p a t o m a (HEP-G2) cells ( 1 1 ) and rat h e p a r o m a (HTC) cells, in w h i c h IGF-I and IGFII also e n h a n c e tyrosine aminotransferase activity via their respective specific r e c e p t o r s ( 1 2 ) . Although studies o f IGF r e c e p t o r s have b e e n r e p o r t e d in bovine (13) and ovine ( 1 4 ) m a m m a r y tissues, and fetal lamb liver m i c r o s o m e s ( 1 5 ) , t h e r e are n o r e p o r t s in the literature on y o u n g adult s h e e p liver IGF receptors. In addition, the interaction o f s h e e p IGF-I and IGF-II w i t h s h e e p liver plasma m e m b r a n e s is e x a m i n e d in this study to d e t e r m i n e if there are distinct species Copyright © 1990 by DOMENDO,INC.
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differences in the receptor binding of homologous ruminant versus nonruminant IGF molecules. During the preparation of this manuscript, a report was published which showed that the ovine IGF molecules differ from human and bovine IGFs by a single amino acid substitution. The sheep IGFs were essentially equipotent with human and bovine IGFs in a variety of immunological and biological tests (16). The current report confirms these findings and extends the results to a homologous ruminant system. The current study was designed to examine the receptor types which exist in postnatal sheep liver plasma membranes in order to provide data on this ruminant species in relationship to the well-studied laboratory animal and human models. In addition, species specificity of IGFs was examined by comparing ovine IGFs with those derived from nonruminant sources. MATERIALS AND METHODS
Recombinant human insulin-like growth factor II (hIGF-II, lot #T51-ZL792) was generously donated by Eli Lilly and Company (Indianapolis, IN). Insulin-like growth factor I (IGF-I, lot "588C) was purchased from Bachem (Torrance, CA). Ovine IGF-I and 1I were purified in our laboratory, as described by Hossner et al. (17). In addition, a chromatofocusing step (18) was employed before HPLC to separate ovine IGF-I and II. Iodogen was purchased from Pierce Chemical Company (Rockford, IL). Bovine serum albumin (BSA) was obtained from Armour Pharmaceutical Company (Kankakee, IL). Aprotinin, ovine insulin, nuclease- and protease-free sucrose and tissue culture grade salts were from Sigma Chemical Company (St. Louis, MO). All other chemicals were reagent grade. M e m b r a n e I s o l a t i o n . Sheep liver membranes were prepared from the hepatic caudate lobe of 2-4 Rambouillet white face range and Suffolk wethers. Approximately 20 g tissue from each wether (100 to 130 lbs; 6-8 months old) was removed 5 to 10 rain postmortem and immediately flushed with ice-cold 1 mM NaHCO 3, 1 mM CaClzcontaining 2 x 105 KIU/I aprotinin (Buffer A). Tissue was minced, homogenized, filtered and centrifuged as described by Ray (19). The upper "fluffy" layer from the low speed centrifugation was suspended in a small volume of Buffer A and combined with sufficient 66% (w/w) icecold sucrose to yield a final concentration of 49% sucrose. The membrane preparation was then divided into four Beckman SW 28.I Ultra-Clear tubes and overlayered with 45% sucrose (13.3 ml), 41% sucrose (10.7 ml), and 37% sucrose (6.5-8.0 ml). Tubes were centrifuged for two hours at 90,000 x g in a Beckman L8-M ultracentrifuge. A plasma membrane fraction was collected at the interface between the 41% and 45% (w/w) sucrose layers and was used in the competitive inhibition assays. Electron microscopy of this fraction showed it to be highly enriched in plasma membranes. Protein content of the membranes was determined by the method of Lowry et al. (20), after hydrolyzing the membranes in 0.5 N NaOH at 37 C for 60 rain. Bovine serum albumin was used as the protein standard. B i n d i n g o f H o r m o n e t o R e c e p t o r s . All peptides were iodinated to a specific activity of 75-210 Ci/g using the Iodogen reagent (21). The iodinated hormone was separated from free iodine on a Sephadex G25 column and repurified immediately before use on a Sephadex G50 column. Studies on membrane dose were performed in duplicate in 1.5 ml Eppendorf microcentrifuge tubes
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in a volume of 225 !11.A HEPES-buffered binding solution (HBS: 100 mM HEPES, 120 mM NaCI, 2.5 mM KCI, 2.5 mM CaCI2, 1.2 mM MgSO4, 1% (w/v) BSA, and 2 x 105 KIU/I Aprotinin, pH 7.4) was used in all studies. Membrane preparations were diluted to desired concentrations with HBS, and sufficient radiolabelled peptide was added to the iced membrane preparation to yield 125,000 c p m / ml. Tubes were then incubated in a shaking water bath (90 opm) at 20 C for 90 rain. After incubation, tubes were centrifuged for one minute in an Eppendorf Model 5414 microcentrifuge. The supernatant was aspirated, and the pellet washed with 100 ttl HBS, followed by a second one minute spin, aspiration, and tip excision. The tip containing the membrane pellet was counted in an Isodata 2 0 / 1 0 Series gamma counter. Specific binding was determined by subtracting the radioactivity bound in the presence of 100 nM hIGF-II from the count obtained from the corresponding incubated sample (total bound). Initial studies were conducted using labelled IGF-I and II with aliquots from each sucrose interface. Further studies were conducted only on t25I-IGF-II binding. Time course and reversibility studies were conducted in duplicate in a volume of 2 ml with 44.4 gg membrane protein. After incubation, duplicate 100 ~tl aliquots were transferred to 1.5 ml Eppendorf microcentrifuge tubes and processed as described above. Reversibility of binding was studied by addition of excess hIGF-II (100 nM) after 90 rain incubation of membranes with ~25I.hIGF-II; also, by centrifugation and resuspension of the membrane pellet in nonradioactive HBS after 90 min incubation with 1251. hIGF-II. Com. petitive inhibition studies were performed with 5 gg plasma membrane protein in 225 gl HBS at 20 C for 90 rain. Binding was examined in the presence of IGF-I, hIGF-II, oIGF-I and II and insulin. C r o s s - l i n k l n g P r o t o c o l s . Purified liver plasma membranes (10 }~g protein per tube) were incubated at 20 C for 90 rain with 5 nM 125I-IGF-II in the absence and presence of 100 nM IGF-I, hIGF-II, oIGF-I and II, and 1,O00 nM ovine insulin. Cross-linking was performed as described in (22). After microcentrifugation to remove nonbound t25I-IGF-II, the membranes were crosslinked with 1 mM disuccinimidyl suberate for 15 rain on ice. E l e c t r o p h o r e s i s a n d A u t o r a d i o g r a p h y . Cross-linked liver plasma membranet25I-IGF-II complexes were electrophoresed by the method of Laemmli (23) on 1.5 mm thick slab gels, with 5% acrylamide separating gels. Samples were reduced with 50 mM dithiothreitol as indicated. Films (Kodak XR-5) were exposed to radioactive gels for three weeks with a Dupont "Cronex" (TM) "Lightning Plus" enhancing screen. Molecular weight standards (Sigma Chemical Co., St. Louis, MO) were: Myosin, 205,000; O-galactosidase, 116,000; bovine serum albumin, 66,000; and ovalbumin, 45,000. RESULTS E v a l u a t i o n o f D i s c o n t i n u o u s S u c r o s e D e n s i t y G r a d i e n t I n t e r f a c e Fractions. Binding of ~25I-IGF-I and II to membranes from the three sucrose density gradient interfaces was evaluated after incubation for 90 min at 20 C. Less than 1.5% specific binding of ~25I-IGF-I was observed with the three interface fractions at 18 to 72 Ixg protein per tube. With 50 ILg of protein per tube, specific binding of t2q-IGF-II by interface fractions 37%-41%, 41%-45%, and 45%-48% was 6.7%, 39.9%, and 26.1%, respectively. The plasma membranes from the 41%-45% sucrose density interface were used for further characterization.
210
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Protein (#g) Fig. 1. Binding of t2q-IGF-II to increasing amounts of purified ovine liver membranes. Membranes (1.6-25 p.g) were incubated for ninety rain at 20 C with 125 I.IGF-II (28,125 cpm) in a total V olume of 225 gl. Nonspecific binding (radioactivity bound in the presence of I00 nM hIGF-II) was subtracted from total radioactivity bound at each membrane dose to determine specific binding. Each point is the mean (_+ SE) of duplicate aliquots of tubes incubated in duplicate. M e m b r a n e D o s e R e s p o n s e . Binding o f x25I-IGF-II to p l a s m a m e m b r a n e s was e v a l u a t e d at p r o t e i n c o n c e n t r a t i o n s o f 1.6-25 llg p e r t u b e (Figure 1). Binding i n c r e a s e d w i t h c o n c e n t r a t i o n u p to 12.5 gg p e r t u b e and was constant t h r o u g h 25 I~g p e r tube. Further studies u s e d 5 ~tg p r o t e i n p e r tube. I n c u b a t i o n T i m e . Liver m e m b r a n e s w e r e i n c u b a t e d w i t h x2sI-IGF-II and total b i n d i n g m e a s u r e d at .25 to 2.5 hr at 20 C. Binding i n c r e a s e d u p to 60 rain i n c u b a t i o n t i m e and p l a t e a u e d t h r o u g h the 2.5 hr i n c u b a t i o n (Figure 2a). N i n e t y m i n u t e s was used as the standard i n c u b a t i o n t i m e for f u r t h e r studies. At 9 C b i n d i n g o f ~2sI-IGF-II i n c r e a s e d w i t h t i m e u p to 12 hr, r e m a i n e d stable t h r o u g h 24 hr a n d d e c r e a s e d b y 22% b y 48 hr o f i n c u b a t i o n (Figure 2b). Reversibility o f Binding. Reversal of 12sI-IGF-II b i n d i n g was evaluated after a d d i n g 100 nM hIGF-II to m e m b r a n e s p r e - i n c u b a t e d for 90 m i n w i t h x2q-IGFII (Figure 3). Fifteen m i n u t e s after a d d i t i o n o f excess IGF-II, b i n d i n g d e c r e a s e d to 92% of c o n t r o l values. At 30 min, b i n d i n g was r e d u c e d b y 18%, w h e r e it r e m a i n e d for the n e x t 75 m i n . The c o n t r o l b o u n d a p p r o x i m a t e l y 44% of a d d e d t25I-IGF-II t h r o u g h o u t the i n c u b a t i o n . Reversibility of b i n d i n g was also s t u d i e d b y r e s u s p e n s i o n of p r e - i n c u b a t e d p l a s m a m e m b r a n e s in n o n r a d i o a c t i v e HBS (Figure 3). Binding was r e d u c e d to 64% of c o n t r o l b y 15 rain after dilution, and r e m a i n e d constant for the r e m a i n i n g i n c u b a t i o n . Total specific b i n d i n g o f the c o n t r o l ( c e n t r i f u g e d and r e s u s p e n d e d in r a d i o a c t i v e HBS) r e m a i n e d constant after r e s u s p e n s i o n . C o m p e t i t i v e I n h i b i t i o n S t u d i e s . C o m p e t i t i v e i n h i b i t i o n studies w e r e perf o r m e d in the p r e s e n c e o f hIGF-I, oIGF-I, hIGF-II, oIGF-II and insulin (Figure 4). The m o s t effective i n h i b i t o r o f 125I-hIGF-II b i n d i n g was hIGF-II, w i t h a h a l f - m a x i m a l i n h i b i t i o n v a l u e of O. 12 nM, f o l l o w e d b y oIGF-II at 1.2 nM. H u m a n IGF-I and oIGF-I w e r e far less p o t e n t inhibitors, w i t h values of 53% B/Bo and 70.9% at 50 nM, respectively. Insulin at 10/~M r e d u c e d b i n d i n g by 13%-
IGF-II BINDING IN SHEEP LIVER
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Af~llntty C r o s s - l t n k i a g Studies. When 125I-IGF-II was incubated with liver plasma membranes, cross-linked with disuccinimidyl suberate, and electrophoresed in the presence of dithiothreitol, a major radioactive band (Mr ----2 7 4 , 0 0 0 ) was observed (Figure 5, lane 1). Coincubation with 100 nM hlGFII (lane 3) or oIGF-II (lane 5) eliminated the radioactive band. There was no effect on the mobility or intensity of the band in the presence of 100 nM hIGF-
212
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In=ulln [MM] Fig. 4. C o m p e t i t i v e i n h i b i t i o n o f ~25I-IGF-II b i n d i n g to liver p l a s m a m e m b r a n e s . M e m b r a n e s (5 lag) w e r e i n c u b a t e d in d u p l i c a t e for 90 rain at 20 C w i t h ~25I-hIGF-II a n d t h e i n d i c a t e d c o n c e n t r a t i o n o f hIGF-II (O), oIGF-II (~k), hIGF-I (O), oIGF-I (/~) or i n s u l i n (11) in a final v o l u m e of 225 }.tl. o Total specific b i n d i n g of ] 2 5 I-IGF-II was 33.6~o. S h o w n are t h e c o m b i n e d results from two experi m e n t s . Each p o i n t r e p r e s e n t s t h e m e a n ( + SE).
IGF-II BINDING IN SHEEP LIVER
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I (lane 2), oIGF-I (lane 4) or 1,000 nM ovine insulin (lane 6). Under nonreducing conditions, the band migrated with an apparent Mr of 210,000215,000 (lane 7). DISCUSSION Although the IGFs are thought to primarily regulate long-term growthassociated changes in the skeleton and muscle (24), recent evidence suggests that IGFs also stimulate short.term glycogen synthesis in hepatocytes (11) and hypoglycemic effects in vivo (25). In addition, IGFs may play a role in hepatic repair mechanisms following injury or partial hepatectomy (26). The significance of these effects and the relative contribution of IGFs to these phenomena are still unknown. The present study demonstrates that purified sheep liver plasma membranes possess abundant, high affinity binding sites for IGF-II. Binding of IGF-II is dependent upon time and temperature of incubation, and membrane concentration. In contrast, very little IGF-I bound to the plasma membranes or other subcellular fractions. Owens et al. (15) demonstrated that fetal sheep liver microsomes contain receptors which interact with rat IGF-II. The current study shows that plasma membranes from young adult wethers also possess Type II, but not Type I, receptors. These observations are similar to those of other investigations which have examined IGF binding to adult rat liver, in which only Type II (IGF-II) receptors are observed (7). The amount of IGF-I binding to the plasma membranes precluded quantitative binding studies with this ligand. Although most studies on normal adult liver tissue have demonstrated the presence of Type
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Fig..5, Autoradiognph of SDS polyacrylamide gel after elecu'ophoresis of ~ZH-hlGF-II crosslinked to sneep dyer plasma membranes. Membranes (10 ~g) were incubated for 90 rain at 20 C with I~I-hlGF-II alone (lane 1) or in the presence of 100 nM hlGF-I (lane 2), hlGF-II (lane 3), olGFI (lane 4), o.IGF-II (lane 5) or 1,0OO nM insulin (lane 6). Samples were crosslinked with 1 mM uisuccinimiayl sunerate and electrnphoresed following reduction with 50 mM dithiothreitol (DTY). The electrophoretic pattern of 12SI-hlGF-lI crosslinked to plasma membranes without DTT reduction is shown in lane 7.
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II receptors, recent studies have shown the existence of small amounts of Type I (IGF-I) receptors in normal rat liver (27) as well as fetal and regenerating rat liver (28). In addition, hepatoma cells contain receptors for IGF-I and IGFII (29). Competitive inhibition studies reported herein also provide evidence that ovine preparations of IGF-I and IGF-II interact with the plasma membranes in a qualitatively similar manner to human recombinant IGF-I and II. Ovine preparations of IGF-I (17) and IGF-II (30) have been reported. The current study shows that the sheep IGFs are less potent inhibitors of 125I-IGF-II binding in competitive binding studies, but we attribute this to differences in purity, of the recombinant and natural sources. Ovine IGF-II, in particular, consists of a mix of po!ypeptides and is an order of magnitude less potent than recombinant IGF-II in inhibiting 125I-IGF-II binding. In the affinity cross- linking studies, specificity of the r e c e p t o r towards IGF-I and II of recombinant or ovine sources was identical. These data provide evidence for the functional identity of ovine IGF-I and II and suggest that species differences between insulin-like growth factors and their receptors are minimal. This has recently been confirmed by studies of the primary sequences of ovine IGF-I and II (16). Affinity cross-linking of a25I-IGF-II to binding sites on the plasma membrane demonstrate the presence of a high molecular weight protein which displays the characte~'istics of a Type II r ecept or (31). Under nonreducing conditions, IGF-II cross-linked to sheep liver plasma membranes migrates with an apparent molecular weight of 2 1 0 , 0 0 0 - 2 1 5 , 0 0 0 and dithiothreitol reduction increases the apparent molecular weight to ~-- 274,000. The observed increase in molecular weight in the presence of a reducing agent is indicative of a single polypeptide chain containing intramolecular disulfide bonds and is consistent with characteristics of the Type II r e c e p t o r in rat liver (32) and human placenta (33). The latter studies estimated the molecular weight of the Type II receptor at 2 20 ,0 0 0 - 2 2 5 ,0 0 0 ( n o n r e d u c e d ) and 255,000-260,000 (reduced). These estimates are similar to our determinations in sheep liver membranes. The observation that adult liver contains predominantly Type II IGF receptors suggests that IGF-I and IGF-II effects are mediated via the Type II receptor or the insulin r e c e p t o r in this tissue. Recently, it has been demonstrated that the Type II r ecep to r binds mannose.6-phosphate in addition to IGF-II (34). As no second messenger for the Type II r e c e p t o r has been identified and studies on its role as a mediator of IGF-II effects are inconclusive (31), the role of this r e c e pto r in IGF physiology is unclear. The current study demonstrates that IGF-II receptors are present in abundance in ruminant liver membranes and that the receptors are similar to those of nonruminant animals.
ACKNOWLEDGEMENTS/FOOTNOTES We thank Dr. R. Bowen for the use of his ultracentrifuge, Dr. A. Tu for sharing his HPLC instrumentation and Eli Lilly & Co. for kindly providing the IGF-II for this study. The excellent technical secretarial skills o f Pat Beebe are gratefully acknowledged. This research was funded by a grant from the United States Department of Agriculture, ~'86-CRCR-l-1943. This is a Loloraoo State University Agriculture Experiment Station Research Publication, project "629.
REFERENCES 1. D a u g h a d a y WH, P h i l l i p s IS, M u e l l e r LC. T h e effects o f i n s u l i n a n d g r o w t h h o r m o n e o n t h e r e l e a s e o f s o m a t o m e d i n b y t h e i s o l a t e d rat liver. E n d o c r i n o l o g y 9 8 : 1 2 1 4 -
1219, 1976. 2. Binoux M, Lassarre C, Hardouin N. Somatomedin production by rat liver in organ culture. III. Studies on the release of insulin-like growth factor and its carrier protein measured by radioligand assays. Effects of growth hormone, insulin, and cortisol. Acta Endocrinol (Copenh) 99:422-430. 1982.
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3. D'Ercole AJ, Applewhite GT, Underwood I.E. Evidence that somatomedin is synthesized by multiple tissues in the fetus. Dev Biol 75:315-328, 1980. 4. Murphy LJ, Bell GI, Friesen HG. Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat. Endocrinology 120:1279-1282, 1987. 5. Nilsson A, Isgaard J, Lindahl A, Dahlstrom A, Skotmer A, Isaksson OGP. Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate. Science 233:571-574, 1986. 6. Schlechter NL, Russell SM, Spencer EM, Nicoll CS. Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin. Proc Natl Acad Sci (USA) 83:7932-7934, 1986. 7. Massague J, Czech MP. The subunit structures of two distinct receptors for insulinlike growth factors I and II and their relationship to the insulin receptor. J Biol Chem 257:5038-5045, 1982. 8. Nissley SP, Rechler MM. Somatomedin/insulin-like growth factor tissue receptors. Clin Endocrinol Metab 13:43-67, 1984. 9. Widmer U, Schmid Ch, Zapf J, Froesch ER. Effects of insulin-like growth factors on chick embryo hepatocytes. Acta Endocrinol 108:237-244, 1985. 10. Parkes JL, Cardell RR, Grieninger G. Insulin-like growth factors (IGF I and IGF II) mimic the effect of insulin on plasma protein synthesis and glycogen deposition in cultured hepatocytes. Biochem Biophys Res Comm 134:427-435, 1986. 11. Verspohl EJ, Maddux EtA, Goldfine ID. Insulin and insulin-like growth factor I regulate the same biological functions in HEP-G2 cells via their own specific receptors. J Clin Endocrinol Metab 67:169- 174, 1988. 12. Heaton JH, Krett NL, Alvarez JM, Gelehrter TD, Romanus JA, Rechler MM. Insulin regulation of insulin-like growth factor action in rat hepatoma cells. J Biol Chem 259:2396-2402, 1984. 13. Dehoff MH, Elgin RG, Collier RJ, Clemmons DR. Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mammary tissue. Endocrinology 122:2412-2417, 1988. 14. Disenhaus C, Belair L, Djiane J. Caracterisation et evolution physiologique des recepteurs pour les < < insulin-like growth factors > > I e t II (IGFs) darts la glande mammaire de brebis. Reprod Nutx Develop 28:241-252, 1988. 15. Owens PC, Waters MJ, Thorburn GD, Brinsmead MW. Insulin-like growth factor receptor in fetal lamb liver: Characterization and developmental changes. Endocrinology 117:982-990, 1985. 16. Francis GL, McNeil KA, Wallace JC, Ballard FJ, Owens PC. Sheep insulin-like growth factors I and II: Sequences, activities and assays. Endocrinology 124:1173-1183, 1989. 17. Hossner KL, Davis SL, Powell C, Sasser RG. Partial purification and characterization of somatomedin from sheep serum. Endocrinology 116:1351-1356, 1985. 18. Blum WF, Ranke MB, Bierich JR. Isolation and characterization of six somatomedinlike peptides from human plasma Cohn fraction IV. Acta Endocrinol 111:271-284, 1986. 19. Ray TK. A modified method for the isolation of the plasma membrane from rat liver. Biochim Biophys Acta 196:1-9, 1970. 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin Phenol Reagent. J Biol Chem 193:265-275, 1951. 21. Fraker PJ, SpeckJC. Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a, 6a-diphenylgly- coluril. Biochem Biophys Res Comm 80:849-857, 1978. 22. Pilch PF, Czech MP. The subunit structure of the high affinity insulin receptor. J Biol Chem 255:1722-1731, 1980. 23. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970. 24. Dodson MY, Allen RE, Shimizu N, Shimizu Y, Hossner KL. Interaction of ovine somatomedin and multiplication stimulating activity/rat insulin-like growth factor II with cultured skeletal muscle satellite cells. Acta Endocrinol (Copenh) 116:425432, 1988.
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25. Guler H-P, ZapfJ, Froesch ER. Short-term metabolic effects of recombinant human insulin-like growth factor I in healthy adults. New England J Med 317:137-140, 1987. 26. Unterman TG, Phillips IS. Circulating somatomedin activity during hepatic regeneration. Endocrinology 119:185-192, 1986. 27. McElduff A, Poronnik P, Baxter RC, Williams P. A comparison of the insulin and insulin-like growth factor I receptors from rat brain and liver. Endocrinology 122:1933-1939, 1988. 28. CaroJF, Poulos J, Ittoop O, Pories wJ, Flickinger EG, Sinha MK. Insulin-like growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver. J Clin Invest 81:976-981, 1988. 29. Heaton JH, Krett NL, Gelehrter TD. Regulation of insulin and insulin-like growth factor (IGF) responsiveness by IGFs in rat hepatoma cells. Endocrinology 118:25552560, 1986. 30. Hey AW, Browne CA, Thorburn GD. Purification and characterization of a fetal somatomedin from sheep: Similarity to insulin-like growth factor II. Endocrinology 122:12-21, 1988. 31. Roth RA. Structure of the receptor for insulin-like growth factor II: The puzzle amplified. Science 239:1269-1271 (1988). 32. Kasuga M, Van Obberghen E, Nissley SP, Rechler MM. Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J Biol Chem 256:53055308, 1981. 33. Massague J, Guillette BJ, Czech MP. Affinity labeling of multiplication stimulating activity receptors in membranes from rat and human tissues. J Biol Chem 256:21222125, 1981. 34. MacDonald RG, Pfeffer SR, Coussens L, Tepper MA, Brocklebank CM, Mole JE, Anderson JK, Chen E, Czech MP, Ullrich A. A single receptor binds both insulinlike growth factor II and mannose-6-phosphate. Science 239:1134-1137, 1988.
Vol. 7(2):207-216, 1990
CHARACTERIZATION OF TYPE II INSULIN-LIKE GROWTH FACTOR (IGF) RECEPTORS IN SHEEP LIVER PLASMA MEMBRANES K. L. Hossner and R. S. Yemm Department of Animal Sciences Colorado State University Fort Collins, Colorado 80523, U.S.A. Received July 21, 1989 ABSTRACT Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of tz~I-hlGF-lI (40%) to liver plasma membranes greatly exceeded that of t251hIGF-I (1.5%) after incubation at 20 C for 90 rain. Binding of Iz~I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of t2SI-hlGF-II was only partially reversed by addition of 1 O0 nM IGF.II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of tzSI-hlGF-II binding demonstrated that IGF.II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 12SI-hIGF-II. Plasma membranes were affinity cross-linked to :25I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,O00-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band..These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type lI receptors. INTRODUCTION Insulin-like g r o w t h factors (IGFs) are g r o w t h - p r o m o t i n g p o l y p e p t i d e s w h i c h are c h e m i c a l l y and b i o l o g i c a l l y related to insulin. Circulating serum IGF's are t h o u g h t to b e p r o d u c e d primarily by the liver (1, 2), although several tissues have b e e n s h o w n to synthesize these p o l y p e p t i d e s (3, 4). G e n e r a l l y c o n s i d e r e d e n d o c r i n e factors, the IGF's also act via a u t o c r i n e a n d / o r paracrine mechanisms (5,6) and r e c e p t o r s for these growth factors are present in many tissues, i n c l u d i n g the liver ('7, 8). Insulin-like g r o w t h factors have b e e n s h o w n to stimulate the i n c o r p o r a t i o n o f glucose into liver cell glycogen, e n h a n c e RNA and p r o t e i n synthesis (9) and e n h a n c e s e c r e t i o n o f a l b u m i n and h e m o p e x i n in c h i c k e m b r y o hepatocytes ( 1 0 ) . Similar effects o n g l y c o g e n synthesis have b e e n s h o w n in h u m a n h e p a t o m a (HEP-G2) cells ( 1 1 ) and rat h e p a r o m a (HTC) cells, in w h i c h IGF-I and IGFII also e n h a n c e tyrosine aminotransferase activity via their respective specific r e c e p t o r s ( 1 2 ) . Although studies o f IGF r e c e p t o r s have b e e n r e p o r t e d in bovine (13) and ovine ( 1 4 ) m a m m a r y tissues, and fetal lamb liver m i c r o s o m e s ( 1 5 ) , t h e r e are n o r e p o r t s in the literature on y o u n g adult s h e e p liver IGF receptors. In addition, the interaction o f s h e e p IGF-I and IGF-II w i t h s h e e p liver plasma m e m b r a n e s is e x a m i n e d in this study to d e t e r m i n e if there are distinct species Copyright © 1990 by DOMENDO,INC.
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differences in the receptor binding of homologous ruminant versus nonruminant IGF molecules. During the preparation of this manuscript, a report was published which showed that the ovine IGF molecules differ from human and bovine IGFs by a single amino acid substitution. The sheep IGFs were essentially equipotent with human and bovine IGFs in a variety of immunological and biological tests (16). The current report confirms these findings and extends the results to a homologous ruminant system. The current study was designed to examine the receptor types which exist in postnatal sheep liver plasma membranes in order to provide data on this ruminant species in relationship to the well-studied laboratory animal and human models. In addition, species specificity of IGFs was examined by comparing ovine IGFs with those derived from nonruminant sources. MATERIALS AND METHODS
Recombinant human insulin-like growth factor II (hIGF-II, lot #T51-ZL792) was generously donated by Eli Lilly and Company (Indianapolis, IN). Insulin-like growth factor I (IGF-I, lot "588C) was purchased from Bachem (Torrance, CA). Ovine IGF-I and 1I were purified in our laboratory, as described by Hossner et al. (17). In addition, a chromatofocusing step (18) was employed before HPLC to separate ovine IGF-I and II. Iodogen was purchased from Pierce Chemical Company (Rockford, IL). Bovine serum albumin (BSA) was obtained from Armour Pharmaceutical Company (Kankakee, IL). Aprotinin, ovine insulin, nuclease- and protease-free sucrose and tissue culture grade salts were from Sigma Chemical Company (St. Louis, MO). All other chemicals were reagent grade. M e m b r a n e I s o l a t i o n . Sheep liver membranes were prepared from the hepatic caudate lobe of 2-4 Rambouillet white face range and Suffolk wethers. Approximately 20 g tissue from each wether (100 to 130 lbs; 6-8 months old) was removed 5 to 10 rain postmortem and immediately flushed with ice-cold 1 mM NaHCO 3, 1 mM CaClzcontaining 2 x 105 KIU/I aprotinin (Buffer A). Tissue was minced, homogenized, filtered and centrifuged as described by Ray (19). The upper "fluffy" layer from the low speed centrifugation was suspended in a small volume of Buffer A and combined with sufficient 66% (w/w) icecold sucrose to yield a final concentration of 49% sucrose. The membrane preparation was then divided into four Beckman SW 28.I Ultra-Clear tubes and overlayered with 45% sucrose (13.3 ml), 41% sucrose (10.7 ml), and 37% sucrose (6.5-8.0 ml). Tubes were centrifuged for two hours at 90,000 x g in a Beckman L8-M ultracentrifuge. A plasma membrane fraction was collected at the interface between the 41% and 45% (w/w) sucrose layers and was used in the competitive inhibition assays. Electron microscopy of this fraction showed it to be highly enriched in plasma membranes. Protein content of the membranes was determined by the method of Lowry et al. (20), after hydrolyzing the membranes in 0.5 N NaOH at 37 C for 60 rain. Bovine serum albumin was used as the protein standard. B i n d i n g o f H o r m o n e t o R e c e p t o r s . All peptides were iodinated to a specific activity of 75-210 Ci/g using the Iodogen reagent (21). The iodinated hormone was separated from free iodine on a Sephadex G25 column and repurified immediately before use on a Sephadex G50 column. Studies on membrane dose were performed in duplicate in 1.5 ml Eppendorf microcentrifuge tubes
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in a volume of 225 !11.A HEPES-buffered binding solution (HBS: 100 mM HEPES, 120 mM NaCI, 2.5 mM KCI, 2.5 mM CaCI2, 1.2 mM MgSO4, 1% (w/v) BSA, and 2 x 105 KIU/I Aprotinin, pH 7.4) was used in all studies. Membrane preparations were diluted to desired concentrations with HBS, and sufficient radiolabelled peptide was added to the iced membrane preparation to yield 125,000 c p m / ml. Tubes were then incubated in a shaking water bath (90 opm) at 20 C for 90 rain. After incubation, tubes were centrifuged for one minute in an Eppendorf Model 5414 microcentrifuge. The supernatant was aspirated, and the pellet washed with 100 ttl HBS, followed by a second one minute spin, aspiration, and tip excision. The tip containing the membrane pellet was counted in an Isodata 2 0 / 1 0 Series gamma counter. Specific binding was determined by subtracting the radioactivity bound in the presence of 100 nM hIGF-II from the count obtained from the corresponding incubated sample (total bound). Initial studies were conducted using labelled IGF-I and II with aliquots from each sucrose interface. Further studies were conducted only on t25I-IGF-II binding. Time course and reversibility studies were conducted in duplicate in a volume of 2 ml with 44.4 gg membrane protein. After incubation, duplicate 100 ~tl aliquots were transferred to 1.5 ml Eppendorf microcentrifuge tubes and processed as described above. Reversibility of binding was studied by addition of excess hIGF-II (100 nM) after 90 rain incubation of membranes with ~25I.hIGF-II; also, by centrifugation and resuspension of the membrane pellet in nonradioactive HBS after 90 min incubation with 1251. hIGF-II. Com. petitive inhibition studies were performed with 5 gg plasma membrane protein in 225 gl HBS at 20 C for 90 rain. Binding was examined in the presence of IGF-I, hIGF-II, oIGF-I and II and insulin. C r o s s - l i n k l n g P r o t o c o l s . Purified liver plasma membranes (10 }~g protein per tube) were incubated at 20 C for 90 rain with 5 nM 125I-IGF-II in the absence and presence of 100 nM IGF-I, hIGF-II, oIGF-I and II, and 1,O00 nM ovine insulin. Cross-linking was performed as described in (22). After microcentrifugation to remove nonbound t25I-IGF-II, the membranes were crosslinked with 1 mM disuccinimidyl suberate for 15 rain on ice. E l e c t r o p h o r e s i s a n d A u t o r a d i o g r a p h y . Cross-linked liver plasma membranet25I-IGF-II complexes were electrophoresed by the method of Laemmli (23) on 1.5 mm thick slab gels, with 5% acrylamide separating gels. Samples were reduced with 50 mM dithiothreitol as indicated. Films (Kodak XR-5) were exposed to radioactive gels for three weeks with a Dupont "Cronex" (TM) "Lightning Plus" enhancing screen. Molecular weight standards (Sigma Chemical Co., St. Louis, MO) were: Myosin, 205,000; O-galactosidase, 116,000; bovine serum albumin, 66,000; and ovalbumin, 45,000. RESULTS E v a l u a t i o n o f D i s c o n t i n u o u s S u c r o s e D e n s i t y G r a d i e n t I n t e r f a c e Fractions. Binding of ~25I-IGF-I and II to membranes from the three sucrose density gradient interfaces was evaluated after incubation for 90 min at 20 C. Less than 1.5% specific binding of ~25I-IGF-I was observed with the three interface fractions at 18 to 72 Ixg protein per tube. With 50 ILg of protein per tube, specific binding of t2q-IGF-II by interface fractions 37%-41%, 41%-45%, and 45%-48% was 6.7%, 39.9%, and 26.1%, respectively. The plasma membranes from the 41%-45% sucrose density interface were used for further characterization.
210
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Protein (#g) Fig. 1. Binding of t2q-IGF-II to increasing amounts of purified ovine liver membranes. Membranes (1.6-25 p.g) were incubated for ninety rain at 20 C with 125 I.IGF-II (28,125 cpm) in a total V olume of 225 gl. Nonspecific binding (radioactivity bound in the presence of I00 nM hIGF-II) was subtracted from total radioactivity bound at each membrane dose to determine specific binding. Each point is the mean (_+ SE) of duplicate aliquots of tubes incubated in duplicate. M e m b r a n e D o s e R e s p o n s e . Binding o f x25I-IGF-II to p l a s m a m e m b r a n e s was e v a l u a t e d at p r o t e i n c o n c e n t r a t i o n s o f 1.6-25 llg p e r t u b e (Figure 1). Binding i n c r e a s e d w i t h c o n c e n t r a t i o n u p to 12.5 gg p e r t u b e and was constant t h r o u g h 25 I~g p e r tube. Further studies u s e d 5 ~tg p r o t e i n p e r tube. I n c u b a t i o n T i m e . Liver m e m b r a n e s w e r e i n c u b a t e d w i t h x2sI-IGF-II and total b i n d i n g m e a s u r e d at .25 to 2.5 hr at 20 C. Binding i n c r e a s e d u p to 60 rain i n c u b a t i o n t i m e and p l a t e a u e d t h r o u g h the 2.5 hr i n c u b a t i o n (Figure 2a). N i n e t y m i n u t e s was used as the standard i n c u b a t i o n t i m e for f u r t h e r studies. At 9 C b i n d i n g o f ~2sI-IGF-II i n c r e a s e d w i t h t i m e u p to 12 hr, r e m a i n e d stable t h r o u g h 24 hr a n d d e c r e a s e d b y 22% b y 48 hr o f i n c u b a t i o n (Figure 2b). Reversibility o f Binding. Reversal of 12sI-IGF-II b i n d i n g was evaluated after a d d i n g 100 nM hIGF-II to m e m b r a n e s p r e - i n c u b a t e d for 90 m i n w i t h x2q-IGFII (Figure 3). Fifteen m i n u t e s after a d d i t i o n o f excess IGF-II, b i n d i n g d e c r e a s e d to 92% of c o n t r o l values. At 30 min, b i n d i n g was r e d u c e d b y 18%, w h e r e it r e m a i n e d for the n e x t 75 m i n . The c o n t r o l b o u n d a p p r o x i m a t e l y 44% of a d d e d t25I-IGF-II t h r o u g h o u t the i n c u b a t i o n . Reversibility of b i n d i n g was also s t u d i e d b y r e s u s p e n s i o n of p r e - i n c u b a t e d p l a s m a m e m b r a n e s in n o n r a d i o a c t i v e HBS (Figure 3). Binding was r e d u c e d to 64% of c o n t r o l b y 15 rain after dilution, and r e m a i n e d constant for the r e m a i n i n g i n c u b a t i o n . Total specific b i n d i n g o f the c o n t r o l ( c e n t r i f u g e d and r e s u s p e n d e d in r a d i o a c t i v e HBS) r e m a i n e d constant after r e s u s p e n s i o n . C o m p e t i t i v e I n h i b i t i o n S t u d i e s . C o m p e t i t i v e i n h i b i t i o n studies w e r e perf o r m e d in the p r e s e n c e o f hIGF-I, oIGF-I, hIGF-II, oIGF-II and insulin (Figure 4). The m o s t effective i n h i b i t o r o f 125I-hIGF-II b i n d i n g was hIGF-II, w i t h a h a l f - m a x i m a l i n h i b i t i o n v a l u e of O. 12 nM, f o l l o w e d b y oIGF-II at 1.2 nM. H u m a n IGF-I and oIGF-I w e r e far less p o t e n t inhibitors, w i t h values of 53% B/Bo and 70.9% at 50 nM, respectively. Insulin at 10/~M r e d u c e d b i n d i n g by 13%-
IGF-II BINDING IN SHEEP LIVER
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Af~llntty C r o s s - l t n k i a g Studies. When 125I-IGF-II was incubated with liver plasma membranes, cross-linked with disuccinimidyl suberate, and electrophoresed in the presence of dithiothreitol, a major radioactive band (Mr ----2 7 4 , 0 0 0 ) was observed (Figure 5, lane 1). Coincubation with 100 nM hlGFII (lane 3) or oIGF-II (lane 5) eliminated the radioactive band. There was no effect on the mobility or intensity of the band in the presence of 100 nM hIGF-
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IGF-II BINDING IN SHEEP LIVER
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I (lane 2), oIGF-I (lane 4) or 1,000 nM ovine insulin (lane 6). Under nonreducing conditions, the band migrated with an apparent Mr of 210,000215,000 (lane 7). DISCUSSION Although the IGFs are thought to primarily regulate long-term growthassociated changes in the skeleton and muscle (24), recent evidence suggests that IGFs also stimulate short.term glycogen synthesis in hepatocytes (11) and hypoglycemic effects in vivo (25). In addition, IGFs may play a role in hepatic repair mechanisms following injury or partial hepatectomy (26). The significance of these effects and the relative contribution of IGFs to these phenomena are still unknown. The present study demonstrates that purified sheep liver plasma membranes possess abundant, high affinity binding sites for IGF-II. Binding of IGF-II is dependent upon time and temperature of incubation, and membrane concentration. In contrast, very little IGF-I bound to the plasma membranes or other subcellular fractions. Owens et al. (15) demonstrated that fetal sheep liver microsomes contain receptors which interact with rat IGF-II. The current study shows that plasma membranes from young adult wethers also possess Type II, but not Type I, receptors. These observations are similar to those of other investigations which have examined IGF binding to adult rat liver, in which only Type II (IGF-II) receptors are observed (7). The amount of IGF-I binding to the plasma membranes precluded quantitative binding studies with this ligand. Although most studies on normal adult liver tissue have demonstrated the presence of Type
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Fig..5, Autoradiognph of SDS polyacrylamide gel after elecu'ophoresis of ~ZH-hlGF-II crosslinked to sneep dyer plasma membranes. Membranes (10 ~g) were incubated for 90 rain at 20 C with I~I-hlGF-II alone (lane 1) or in the presence of 100 nM hlGF-I (lane 2), hlGF-II (lane 3), olGFI (lane 4), o.IGF-II (lane 5) or 1,0OO nM insulin (lane 6). Samples were crosslinked with 1 mM uisuccinimiayl sunerate and electrnphoresed following reduction with 50 mM dithiothreitol (DTY). The electrophoretic pattern of 12SI-hlGF-lI crosslinked to plasma membranes without DTT reduction is shown in lane 7.
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II receptors, recent studies have shown the existence of small amounts of Type I (IGF-I) receptors in normal rat liver (27) as well as fetal and regenerating rat liver (28). In addition, hepatoma cells contain receptors for IGF-I and IGFII (29). Competitive inhibition studies reported herein also provide evidence that ovine preparations of IGF-I and IGF-II interact with the plasma membranes in a qualitatively similar manner to human recombinant IGF-I and II. Ovine preparations of IGF-I (17) and IGF-II (30) have been reported. The current study shows that the sheep IGFs are less potent inhibitors of 125I-IGF-II binding in competitive binding studies, but we attribute this to differences in purity, of the recombinant and natural sources. Ovine IGF-II, in particular, consists of a mix of po!ypeptides and is an order of magnitude less potent than recombinant IGF-II in inhibiting 125I-IGF-II binding. In the affinity cross- linking studies, specificity of the r e c e p t o r towards IGF-I and II of recombinant or ovine sources was identical. These data provide evidence for the functional identity of ovine IGF-I and II and suggest that species differences between insulin-like growth factors and their receptors are minimal. This has recently been confirmed by studies of the primary sequences of ovine IGF-I and II (16). Affinity cross-linking of a25I-IGF-II to binding sites on the plasma membrane demonstrate the presence of a high molecular weight protein which displays the characte~'istics of a Type II r ecept or (31). Under nonreducing conditions, IGF-II cross-linked to sheep liver plasma membranes migrates with an apparent molecular weight of 2 1 0 , 0 0 0 - 2 1 5 , 0 0 0 and dithiothreitol reduction increases the apparent molecular weight to ~-- 274,000. The observed increase in molecular weight in the presence of a reducing agent is indicative of a single polypeptide chain containing intramolecular disulfide bonds and is consistent with characteristics of the Type II r e c e p t o r in rat liver (32) and human placenta (33). The latter studies estimated the molecular weight of the Type II receptor at 2 20 ,0 0 0 - 2 2 5 ,0 0 0 ( n o n r e d u c e d ) and 255,000-260,000 (reduced). These estimates are similar to our determinations in sheep liver membranes. The observation that adult liver contains predominantly Type II IGF receptors suggests that IGF-I and IGF-II effects are mediated via the Type II receptor or the insulin r e c e p t o r in this tissue. Recently, it has been demonstrated that the Type II r ecep to r binds mannose.6-phosphate in addition to IGF-II (34). As no second messenger for the Type II r e c e p t o r has been identified and studies on its role as a mediator of IGF-II effects are inconclusive (31), the role of this r e c e pto r in IGF physiology is unclear. The current study demonstrates that IGF-II receptors are present in abundance in ruminant liver membranes and that the receptors are similar to those of nonruminant animals.
ACKNOWLEDGEMENTS/FOOTNOTES We thank Dr. R. Bowen for the use of his ultracentrifuge, Dr. A. Tu for sharing his HPLC instrumentation and Eli Lilly & Co. for kindly providing the IGF-II for this study. The excellent technical secretarial skills o f Pat Beebe are gratefully acknowledged. This research was funded by a grant from the United States Department of Agriculture, ~'86-CRCR-l-1943. This is a Loloraoo State University Agriculture Experiment Station Research Publication, project "629.
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