Insulin receptors in lizard brain and liver: structural and functional studies of α and β subunits demonstrate evolutionary conservation

Diabetologia, May 1986

Summary Specific insulin receptors are present in the liver and brain of the lizard Anolis carolinesis. In this study, the specific binding of 125I-insulin to the receptors showed time, temperature and pH dependency. Specific binding to crude membranes prepared from brain was 1–2% of the total radioactivity added compared to 4–5% in the crude membranes prepared from liver. Solubilization and wheat germ agglutinin purification of the membranes resulted in an increase in the specific binding (per mg of protein) between 6 and 32 times for liver membranes and 13–186 for brain membranes. Binding inhibition of tracer insulin by unlabeled porcine insulin was characteristic for insulin receptors with 50% inhibition for liver crude membranes at 60 ng/ml of porcine insulin and 0.7 ng/ml for purified brain insulin receptors. Chicken insulin was 2- to 3-fold more potent and proinsulin about 100 times less potent than porcine insulin. The α-subunits of liver and brain had apparent molecular weights on sodium dodecyl sulfate polyacrylamide gel electrophoresis of 135 kDa and 120 kDa respectively. Apparent molecular weights of β subunits were 92 kDa for both tissues. Insulin stimulated phosphorylation of the β subunit of both brain and liver receptors. Both tissues demonstrated tyrosine-specific phosphorylation, which was stimulated by insulin, of exogenously added artificial substrates. In addition, purified brain insulin receptor preparations contained an endogenous protein with apparent molecular weight of 105 kDa, whose phosphorylation was stimulated by insulin (10−7 mol/l). This phosphoprotein was not immunoprecipitated by anti-insulin receptor antibodies. These studies suggest that the structural differences between brain and liver receptors previously demonstrated in the rat are also present in the lizard, which is about 300,000,000 years older than the mammalian species. Thus, there is strong evolutionary conservation of the brain insulin receptor.

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Insulin receptors in lizard brain and liver: structural and functional studies of α and β subunits demonstrate evolutionary conservation

Diabetologia Insulin receptors in lizard brain and liver: structural and functional studies o f a and fl subunits demonstrate evolutionary conservation J. Shemer 0 J. C. Penhos 0 D. LeRoith 0 0 Sectionof Molecularand Cellular Physiology,DiabetesBranch,National Institute of Arthritis, Diabetes , Digestiveand KidneyDiseases, RockvillePike,Bethesda,Maryland , USA Summary. Specific insulin receptors are present in the liver and brain of the lizard Anolis carolinesis. In this study, the specific binding of 12sI-insulin to the receptors showed time, temperature and pH dependency. Specific binding to crude membranes prepared from brain was 1-2% of the total radioactivity added compared to 4-5% in the crude membranes prepared from liver. Solubilization and wheat germ agglutinin purification of the membranes resulted in an increase in the specific binding (per mg of protein) between 6 and 32 times for liver membranes and 13-186 for brain membranes. Binding inhibition of tracer insulin by unlabeled porcine insulin was characteristic for insulin receptors with 50% inhibition for liver crude membranes at 60 ng/ml of porcine insulin and 0.7 ng/mt for purified brain insulin receptors. Chicken insulin was 2- to 3-fold more potent and proinsulin about 100times less potent than porcine insulin. The a-subunits of liver and brain had apparent molecular weights on sodium dodecyl sulfate polyacrylamide gel electrophoresis of 135kDa and Insulin receptor; nervous system; phosphorylation; evolution; reptiles - The insulin receptor is a membrane b o u n d glycoprotein composed of two a-subunits and two fl subunits joined by disulfide bonds [ 1 ]. The recent cloning of the insulin receptor gene and the derivation of the amino acid sequence from the gene has suggested that the a subunit is predominantly an extracellular subunit containing most of the glycosylation sites. The /3 subunit contains a transmembrane domain as well as a cytoplasmic domain with the tyrosine phosphorylation site [ 2, 3 ]. Binding of insulin to the insulin receptor occurs primarily through the a subunit, and the/3 subunit is capable of autophosphorylation as well as tyrosine specific phosphorylation of exogenous substrates [ 4, 5 ], a function which it shares with other growth factors such as epidermal growth factor, insulin-like growth factor I, and platelet derived growth factor [6]. Insulin receptors are found on most tissues in all mammalian and vertebrate species studied [ 7-17 ]. Although a specific function for insulin on the nervous 120 kDa respectively. Apparent molecular weights of fl subunits were 92 kDa for both tissues. Insulin stimulated phosphorylation of the fl subunit of both brain and liver receptors. Both tissttes demonstrated tyrosine-specific phosphorylation, which was stimulated by insulin, of exogenously added artificial substrates. In addition, purified brain insulin receptor preparations contained an endogenous protein with apparent molecular weight of 105 kDa, whose phosphorylation was stimulated by insulin (10-7 tool/l). This phosphoprotein was not immunoprecipitated by anti-insulin receptor antibodies. These studies suggest that the structural differences between brain and liver receptors previously demonstrated in the rat are also present in the lizard, which is about 300,000,000years older than the mammalian species. Thus, there is strong evolutionary conservation of the brain insulin receptor. system has not been convincingly demonstrated, the wide distribution of the insulin receptor on specific cell types in the nervous system strongly suggests that insulin may play an important role in the physiology of the nervous system [11, 17, 181. Recently, structural and functional studies of the insulin receptor in the rat brain have suggested that the a subunit of rat brain insulin receptor is smaller than that in non-neural tissues such as liver and adipocytes [ 19-22 ]. Despite these structural changes the rat brain insulin receptor is functional, demonstrating phosphorylation of the fl subunit and tyrosine specific phosphorylation of artificially synthesized exogenous substrates [23]. This difference in structure has been assigned to a variation in the carbohydrate moeities in the rat brain insulin receptor. To determine whether the difference in brain and non-neural insulin receptors of the rat is species-specific and whether this is evolutionarily well conserved, we have studied Anolis carolinesis (lizard), a representative o f the reptile family. In this study we demonstrate that the brain insulin receptor of the lizard differs from liver receptors similar to the differences found in the rat. Thus, the a subunit of the lizard brain insulin receptor has a smaller molecular weight than that o f the liver receptor. Despite this structural difference, the fl subunit of both brain and liver receptors demonstrate insulindependent autophosphorylation and tyrosine specific phosphorylation of exogenous substrates. Materials and methods Materials 125I-insulin (specific activity 280-370 mCi/mg) and [y-32p]ATP (specific activity 2900 Ci/mol) were purchased from New England Nudear (Boston, MA, USA). Porcine insulin was purchased from Elanco (Indianapolis, IN, USA) and biosynthetic human proinsulin (A18-4U6-253) was purchased from Eli Lilly and Co (Indianapolis, IN, USA). Chicken insulin (from Littron Laboratories) was obtained through the Research Resources Program of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health (Bethesda, MD, USA). Wheat germ agglutinin (WGA) coupled to agarose was purchased from Miles-Yeda Ltd. (Rehovot, Israel). Phenylmethylsulfonyl Fluoride (PMSF), Bacitracin, Leupeptin and Aprotinin were purchased from Sigma Chemical (St. Louis, MO, USA). Bovine serum albumin (insulin-free) was purchased from Armour Pharmaceutical (Kankakee, IL, USA). Guinea pig anti-insulin antiserum (batch 625) was purchased from the Department of Pharmacology, Indiana University (Indianapolis, IN, USA). Patient sera containing anti-insulin receptor antibodies (designated B8, B10) and anti-pp120 antibodies were generously donated by Dr. Simeon Taylor, National Institutes of Health, (Bethesda, MD, USA). CTP, ATP, N-acetyl-D-glucosamine, neuraminidase (from Clostridium perfringens, type N2133), and artificial tyrosine containing substrates, i.e. poly (Glu, Tyr), 4:1, poly (Glu, Tyr), 1:1, poly (Glu, Ala, Tyr), 6: 3 : 1, poly (Glu, Ala, Tyr), 1 : 1 :1, poly (Glu, Ala, Tyr), 6: 3 :1 and poly (Ala, Gin, Lys, Tyr), 6 :2: 5 : 1 were purchased from Sigma Chemical (St. Louis, MO, USA). All reagents for electrophoresis were obtained from Bio-Rad Laboratories (Richmond, CA, USA). Protein A (pansorbin) was purchased from Calbiochem Behring (La Jolla, CA, USA). Preparation o f crude membrane Male adult lizards, Anolis carolinesis (1.5-4.5g), were purchased from Carolina Biological Supply Co. (Burlington, NC, USA). The animals were decapitated; the brains and livers were immersed in icecold homogenization buffer (15 v/w) containing 1 mmol/1 NaHCO3, 2 mmol/1 PMSF, 10 ~tg/mi Leupeptin and Aprotinin at a final concentration of I trypsin inhibitory unit per ml. Tissues were homogenized in a glass/glass homogenizer (Ten Brock Model, Coming Glass Works, Coming, NY, USA) using 20 strokes of the pestle. The homogenate was centrifuged at 600 g f o r 10 min at 4 °C. The resultant supernatant was centrifuged at 20,000 g for 30 min at 4 °C and the supernatant was discarded. The pellet was then resuspended in KRP assay buffer (Krebs Ringer phosphate buffer, Ca ++ free, containing 150mmol/1 NaC1, 5 mmol/1 KC1, 1.2mmol/1 KH2PO4, 16mmol/1 potassium phosphate buffer, 16retool/1 NazHPO4 and 1.2 mmol/1 MgSO4.7H20) to a protein concentration of approximately 10 mg/ml as determined by the Lowry method [ 24 ] and stored at - 70 °C in aliquots for up to 6 months before use. Insulin binding studies Binding assays were performed as previously described by Havrankova et al. [ 11 ]. Time dependency, optimal pH and protein concentration were determined. For competition binding assays, the final membrane protein concentration was 700 ~tg/ml for fiver and brain tissues. Total reaction volume of the assay was 150 ~tl KRP buffer consisting of 12sIinsulin (0.3 ng/ml), 1% bovine serum albumin and I mg/ml Bacitracin, with or without unlabeled pork insulin (final concentration 0-105 ng/ml), and included 50 ~tl of membranes, Specificity studies included the use of proinsulin and chicken insulin. Radioactivity bound to the membranes in the presence of 10 ~tg/ml of unlabeled pork insulin was designated "nonspecific binding" and was subtracted from the total binding to obtain "specific binding". The assays were performed in microfuge tubes at 4 °C for 18 h. After incubation, the tubes were centrifuged for 3 min; the supernatants were aspirated and discarded. The pellets were washed with KRP buffer containing 0.5 mol/1 sucrose; the microfuges were spun again for 3 min, the supernatants were aspirated and the tips of the microfuge tubes containing the pellets were counted in a ~/counter. Degradation of 125I-insulin was determined by its solubility in 5% trichloroacetic acid and was found to be negligible (< 5% after 18 h at 4 °C). Solubilization of the membrane and wheat-germ agglutinin chromatography Both liver and brain membranes were solubilized as previously described by Hedo et al. [ 25 ]. Briefly, the pellets of the crude membranes were resuspended in 50 mmol/1 Hepes buffer (pH 7.8) with 1% Triton X-100 and 2mmol/1 PMSF. The homogenate was centrifuged at 40,000 g for 45 min at 4 °C and the supernatant with the solubilized receptors was saved. 30-240mg solubilized membrane protein from brain and 30 mg of liver were affinity purified in a volume of 7 ml buffer over a 2-ml WGA column. The columns were etuted as 1-ml fractions with 7-20 ml of the same washing buffer containing 0.3 mol/1 Nacetyl-D-glucosamine. The protein concentration [ 26 ], as well as tracer binding (see below), were determined in aU fractions. The fractions with the highest protein concentration and the highest specific binding (10-20%) were stored at - 7 0 °C for up to 6 months before u s e . Solubilized receptor binding assays Binding assays were performed as previously described by Harrison and Itin [ 27 ] with minor modifications. Briefly, the total reaction volume of the assay was 200 ~tl,and consisted of 50 ~tl of the assay buffer at pH 7.8 (50 mmol/1 Hepes, 150 retool/1 NaC1 and 0.1% BSA), 125 ~tl of 12sI-insulin diluted in the assay buffer (final concentration 0.3 n g / ml) with 1 mg/ml Bacitracin, 0.1% BSA and 25 ~tl of the solubilized and WGA purified membranes (-160 ~tg/ml) in 0.1% Triton X-100. The final membrane protein concentration in the incubation was 20 lxg/ml, i. e. 4 ~tgof membrane protein per assay tube. The assay was incubated for 4 h (as indicated by the time course, Fig.3) with and without pork insulin, proinsulin and chicken insulin (final concentration 0-105 ng/ml), at 22° -24°C. To terminate the reaction, 100 p~lof 0.3% bovine y-globulin and 300 ~tl of 25% polyethylene glycol was added and the tubes immediately chilled. Precipitates were collected by centrifugation at 2500 g for 15 rain at 4 °C, and washed once with 300 ~tl of 12.5% polyethylene glycol. The supernatants were aspirated and discarded and the radioactivity in the tubes counted in the y counter. Radioactivity bound to the membranes in the presence of 10 p.g/ml of unlabeled pork insulin was designated "non-specific binding" and subtracted from the total binding to obtain "specific binding". Degradation of 125I-insulin was determined by precipitation with 5% trichloroacetic acid and was found to be negligible (< 5% after 4 h at 22° -24 °C). Crosslinking o f 125I-inSulin to insulin receptors Crosslinking of 125I-insulin to insulin receptors was performed as described previously by Taylor et al. [ 28 ] with minor modifications. Lizard liver or brain membranes [final concentration 10 and 20 mg/ml re-fold stimulation 1.5 1.3 1.3 Brain and liver insulin receptor preparations containing 4-10 ~tgof protein were incubated in the presence or absence of 10 -7 mol/l porcine insulin at 22° -24°C for 30 min. Phosphorylation was performed for 30 min. In repeat experiments values for -fold stimulation using poly(Glu, Tyr) 4:1 gave results of 5-to 10-fold for brain and 1.5-2.0 for liver. Using similar conditions for rat tissues, -fold stimulation was 1.7 and 5.0 for brain and liver respectively spectively] were added to 125I-insulin [final concentration 10ng/ml] and incubated overnight at 4°C. Sprague-Dawley rat liver and brain membranes (final concentration 5 mg/ml) were also studied for comparison. Incubations were terminated by centrifugation at 12,000 g for 30 min at 4 °C. The pellets were resuspended in and washed twice with BSA-free KRP pH 7.8. Crosslinking of receptor was performed in KRP pH 7.8 in the presence of 0.1 mmol/1 disuccinimidyl suberate (DSS) for 30 min on ice. The crosslinking was terminated by the addition of 100 mmol/1Tris with 10 mmol/1 EDTA pH 7.4 and centrifugation at 12,000 g for 10 rain at 4°C. Supernatants were aspirated and pellets were resuspended in 50 mmol/1 Hepcs pH 7.8. Solubilization of membranes was performed in 1% Triton X-100 and 2mmol/1 PMSF for 18 h at 4°C. The solubilized membranes were ultracentrifuged at 45,000 g for 45 rain at 4°C and the supernatants collected. Immunoprecipitation was carried out by adding 10 txl of guinea pig anti-insulin antiserum (final dilution 1:100) for 18 h at 4°(;. The immune complexes that bound to Pansorbin were sedimented by centrifugation at 12,000 g for 5 rain. The pellets were washed twice with 50 mmol/1 Hepes buffer pH 7.8 containing 0.1% Triton X-100. After the addition of 110 111sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer the tubes were vortexed and heated at 95 °C for 10 min. The Pansorbin was sedimentated by centrifugation, and 50 Ixl of the supernatant containing the crosslinked receptors were subjected to SDS-PAGE. Autoradiography of the dried slab gel was carried out for 3 days. Neuraminidase digestion o f erosslinked receptor Either liver or brain receptors were crosslinked to 125I-insulin as described above. Prior to solubilizationpellets were resuspended in I ml of a solution of 5 m m o l / l 2(N-morpholino) ethanesulfonic acid (MES) and I mmol/1 CaC12,pH 6, in the presence or absence of 2.5 U of neuraminidase (type VIII) from Clostridium perfringens and incubated for 30 min at 37 °C [ 22 ]. The pellets were washed once in KRP pH 7.8, and solubilized, immunoprecipitated and subjected to SDSPAGE as described above. Phosphorylation o f artificial substrates Phosphorylation of artificial substrates by WGA purified receptors was performed as described by Rees-Jones et al. [ 23 ]. Briefly, 40 i.tlof WGA purified receptors containing 4-10 11gof protein were incubated (30 min at 22 °C-24 °C) in total volume of 140 111buffer (60 mmol/1 Hepes, 50 mmol/1 NaC1, 0.03% Triton X-100, 0.014% BSA pH 7.6) and 400 txg of various artificial substrates (see Table 1) in the presence or absence of various concentrations of insulin and proinsulin (10-9 to 10-6 mol/1). Phosphorylation was initiated by addition of reaction mix to yield a final concentration of 50 11moi/1 (~/-32p]ATP(specific activity - 3 11Ci/nM), I mmol/1 CTP and 20 mmol/l MgCI2. After 5, 15 and 30rain at 20° -24 °C, 75-tll samples were spotted on squares of Whatmann 3MM filter paper and 30 s later were placed in 10% trichloroacetic acid solution, 10mmol/1 Na pyrophosphate, then washed extensively. Filter papers were dried and 32p incorporation was determined by counting in a liquid scintilation counter. Insulin receptor autophosphorylation Insulin receptor autophospborylation and immunoprecipitation of the phosphorylated receptors were performed as previously described [ 5, 29 ]. Forty microliters WGA-purified receptor preparation were incubated (30min at 24°C) in a total volume of 140pJ of buffer (60mmol/1 Hepes, 50mmol/1 NaC1, 0.03% Triton X-100, 0.014% BSA, pH 7.6) in the presence or absence of 10-7mol/1 insulin. Phosphorylation was initiated by adding 40 ~tlof reaction mix to yield a final concentration of 50 11mot/1[7-32p]ATP(specific activity N 3 11Ci/ nmol), 1 mmol/1 CTP, 3 mmol/1 manganese acetate pH 7.6. After 5 min at 22 °C-24 °C the reaction was terminated by adding 50 11mol/1 of 2.5-fold concentrated SDS-PAGE sample buffer to each 75 111of assay samples. The samples were heated for 10 rain at 95 °C and analyzed by SDS-PAGE and autoradiography. Immunoprecipitation of the phosphorylated receptors was performed by increasing the volume of the phosphorylation assay mixture 2-fold, decreasing [7-32p]ATP to 5 11mol/1 final concentration (specific activity ~ 50 IxCi/nmol). Termination of phosphorylation was achieved by adding one-third volume of "stopping solution" containing Triton X-100 (0.2% v/v), EDTA (10 mmol/l), NaF (0.1 mol/1), sodium pyrophosphate (20 mmol/1), sodium phosphate (20 mmol/1), ATP (20 mmol/1), Tris base (0.25 mol/l), SDS (5% v/v), glycerol (25% v/v), 2-mercaptoethanol (1.82 mol/1) and bromphenol blue (0.02% v/v), pH 7.6. Antiserum B-8 and B-10 (containing anti-insulinreceptor antibodies), rabbit control serum at I : 100 dilution, and anti pp 120 antiserum at I :40 dilution were then added. The washed immunoprecipitates were boiled in 60 111of SDS-PAGE sample buffer and analysed by SDS-PAGE and autoradiography. Results Insulin binding to liver membranes T h e s p e c i f i c b i n d i n g o f 1 2 5 I - i n s u l i n t o l i v e r c r u d e m e m b r a n e w a s 4 t o 5 % o f t h e t o t a l r a d i o a c t i v i t y a d d e d u s i n g 700/p~g m e m b r a n e p r o t e i n / m l as f i n a l c o n c e n t r a t i o n a n d w a s d e p e n d e n t o n t i m e , t e m p e r a t u r e ( F i g . 1), p H , a n d p r o t e i n c o n c e n t r a t i o n ( d a t a n o t s h o w n ) . M a x i m a l ~, 2 -3 ,'7 o~ _z J 1 D co z 7 5 TIME (h) .5 1,5 2 3 6 ' 1 1 Fig.l. Time course of association of 125I-insulin with lizard liver crude membranes. Membranes were prepared from lizard liver homogenates using 1 mmol/1 NaHC03 and protease inhibitors, and were incubated at the various temperatures in KRP buffer (pH 7.8) at a final protein concentration of 700 ~tg/ml. At the indicated time, the reaction was stopped by centrifugation of the tubes for 3 ntin. The a25Iinsulin bound to the membranes in the presence of 10 Fg/ml unlabeled insulin was considered non-specific and was subtracted from the total binding to give specific insulin binding. Specific binding is expressed as percent of total counts added using 700 txg membrane protein/ml (final concentration). 4 °C ( • ) , 15 °C (O), 22 °C (zx) and 37 °C (A). Non-specific binding was 25% of the total binding at 4°C, 44% at 15 °C, 83% at 22 °C and nearly 100% at 37 °C at 4 h 1 10 100 POLYPEPTtDE (ng/ml) 1000 10,000 Fig.2. Competition of 125I-insulinbinding to lizard liver crude membranes by insulin analogs. Crude membranes were incubated at 4 °C for 18 h at a final protein concentration of 700 p,g/ml in KRP buffer (pH 7.8) with 125I-insulin(0.3 ng/ml) together with various concentrations (0-10 ~tg/ml) of porcine insulin ( 0 ) proinsulin ( • ) and chicken insulin (O) from 0 to 10~tg/rnl. The incubation was stopped by centrifugation of the tubes at 4 °C for 3 min and specific binding determined. Fifty percent inhibition of binding was achieved in the presence of 60 ng/ml of porcine insulin. The relative potencies were chicken insulin (@) > porcine insulin (O) > proinsulin ( • ) s p e c i f i c b i n d i n g w a s a c h i e v e d a t 4 °C a f t e r 4 - 1 8 h, a n d t h e o p t i m a l p H w a s 7.8. T h e o p t i m a l f i n a l p r o t e i n c o n c e n t r a t i o n w a s 0.5 t o 0.85 m g / m l . B i n d i n g i n h i b i t i o n o f 125I-insulin b y u n l a b e l e d p o r c i n e i n s u l i n w a s t y p i c a l f o r i n s u l i n r e c e p t o r s ( F i g . 2), w i t h 50% i n h i b i t i o n at 60 n g / m l . T h e l i v e r r e c e p t o r s d i s p l a y e d t h e s p e c i f i c i t y c h a r a c t e r i s t i c o f i n s u l i n r e c e p t o r s w i t h t h e a f f i n i t y f o r c h i c k e n i n s u l i n > p o r c i n e > p r o i n s u l i n . U s i n g t h e s a m e t e c h n i q u e s , t h e s p e c i f i c b i n d i n g o f 125I-insulin t o r a t l i v e r m e m b r a n e s w a s 7 - 8 % o f t h e t o t a l r a d i o a c t i v i t y a d d e d . ~5 "5 Q4 Fig.4. Competition of 125I-insulinbinding by insulin analogs to solubilized and WGA purified insulin receptor from lizard brain. 125I-insulin (0.3ng/ml) was incubated at 22°C in 200 ~tl Hepes buffer (50 mmol/1) pH 7.8 containing 0.15 mol/1 NaC1 0.1% BSA and I mg/ ml Bacitracin with solubilized and partially purified brain receptor in a final protein concentration of 20 ~tg/ml. Porcine insulin (O), chicken insulin ( 0 ) and proinsulin (A) were used at various concentrations from 0-10 p~g/ml.The incubation was stopped by adding 100 ~tl of 0.3% bovine 7/-globulin and 300 pAof 25% polyethylene glycol. After 2 washes the tubes with the precipitate were counted in a y counter and the specific binding determined. Fifty percent inhibition binding was achieved with 0.7 ng/ml of porcine insulin. The relative potencies were chicken insulin (@) > porcine insulin ( 0 ) > proinsulin ( A ) 11 10987Fig. 5. Elufion profile of lizard liver and brain insulin receptors from WGA columns. Separate WGA columns (2 ml of settled gel) were loaded with 7 ml of solubilized liver membranes containing 30 nag of protein (upper panel) and 21 ml of solubilized brain membranes containing 90 mg of protein (lower panel). The insulin receptors were eluted with linear gradients (9 ml) of N-acetyl-D-glucosamine as I ml fractions. The protein concentration was determined using fluorescamine [ 26 ] and the a25I-insulin specifically bound calculated as a percentage of the total counts added. The lizard brain insulin receptors demonstrated an unusual elution profile (lower panel). The specific binding of 125I-insulin remained high (N 9%) despite the fall of protein concentration from 500 Ixg/ml (fraction 2) to 25 sxg/ml (fraction 9). A similar profile for brain insulin receptors was obtained after application of 7 ml of solubilized membranes containing 30 mg of protein Insulin binding to brain membranes M a x i m a l specific b i n d i n g o f 125I-insulin to brain crude m e m b r a n e s w a s 1.2% o f total radioactivity a d d e d u s i n g 700-~tg m e m b r a n e p r o t e i n p r o t e i n / m l as final c o n c e n trations b y 1 8 h at 4 ° C , p H 7.8 w i t h 50% i n h i b i t i o n at 4.0 n g / m l (data n o t s h o w n ) . T h e specific b i n d i n g w a s a l s o time, p H , t e m p e r a t u r e a n d p r o t e i n c o n c e n t r a t i o n d e p e n d e n t . H o w e v e r , d u e to l o w specific b i n d i n g o f brain crude m e m b r a n e s , detailed data for these p a r a m e ters w e r e s t u d i e d f o l l o w i n g W G A purification (see below). Specific b i n d i n g to rat brain m e m b r a n e s w a s 5-6%. Insulin binding to solubilized and wheat germ agglutinin purified brain receptors F o l l o w i n g W G A purification o f brain m e m b r a n e s (see b e l o w ) , specific b i n d i n g o f 125I-insulin w a s t i m e a n d t e m p e r a t u r e d e p e n d e n t (Fig.3). T h e s o l u b i l i z e d a n d W G A - p u r i f i e d brain receptors e x h i b i t e d 50% inhibition Fig.6. SDS polyacrylamide gel electrophoresis of 125I-insulin crosslinked to liver and brain insulin receptors and the effect of neuraminidase. Lizardbrain and liver insulin receptors were crosslinked, immunoprecipitated and treated with neuraminidase. Sprague-Dawley ratbrain and liver membranes were also studied for comparison. Lane A-brain receptors, incubated with excess of unlabeled insulin, lane Bcrosslinked lizard brain receptors, lane C-crosslinked lizard brain receptors after neuraminidase digestion. Lane E-lizard liver receptors incubated with excess of unlabeled insulin, lane F-crosslinked lizard liver receptors, lane G-lizard liver receptors after neuraminidase digestion. The apparent molecular weight of the a subunit of the lizard liver receptor is 135 kDa similar to rat liver (lane H) and decreased to 120kDa after neuraminidase digestion. The apparent molecular weight of the lizard brain subunit is 120 kDa, similarto rat brain (lane D) and is not affected by neuraminidase digestion. The exact nature of the higher molecular weight bands present in lanes F and G is not known. They may represent partial degradation of the insulin receptor oligomer, since excess cold insulin displaces the label and neuraminidase affects the molecular weight to a similar degree when compared to the a-subunit of t25I-insulin in the presence of 0.7 ng/ml unlabeled pork insulin, and displayed the specificity characteristic of insulin receptors with the affinity for chicken insulin > porcine insulin > proinsulin (Fig. 4). Wheat germ agglutinin chromatography o f the solubilized liver and brain membranes W G A c h r o m a t o g r a p h y o f the s o l u b i l i z e d liver a n d brain m e m b r a n e s in 1% Triton X - 1 0 0 resulted in an increase in total specific b i n d i n g per m g protein. T h e -fold increase w a s 6 to 32 t i m e s for liver m e m b r a n e s a n d 13 to 186 times for the brain m e m b r a n e s ( d e p e n d i n g o n the fraction number, Fig. 5). T h e e l u t i o n profile o f the insulin receptor f r o m W G A c o l u m n s w a s different in the t w o tissues (Fig. 5). T h e specific b i n d i n g o f 125I-insulin to brain receptor remained high and constant despite the fall in protein concentration from 500 ~xg/ml (fraction 2) to 25 txg/ml (fraction 9). Cross-linking of l2sI-insulin to liver and brain receptors Crosslinking of the insulin receptor and SDS-PAGE run under reducing conditions revealed a subunits from both brain and liver insulin receptors (Fig. 6, lane B and F). The brain receptor c¢subunit had an apparent MW of 120 kDa compared to 135 kDa for liver. Unlabeled insulin (10 p~g/ml) abolished the bands from both brain and liver (Fig. 6, lanes A and E). Neuraminidase treatment increased the electrophoretic mobility of the a subunit from liver (Fig. 6, lane G) but failed to affect brain a subunit (Fig. 6, lane C). Phosphorylation of exogenous substrates by liver and brain insulin receptors Insulin stimulated the receptor-induced phosphorylation of synthetic tyrosine polymers (Table 1). This effect was time and dose dependent (data not shown) with maximum effect at an insulin concentration of 10-7moi/1. The most efficient incorporation was demonstrated using poly (Glu, Tyr), 4:1. Basal incorporation of 32p was high using liver receptors. Insulin stimulation was 5-10times basal using brain receptor and 1.5-2 times using liver receptors. No incorporation was seen using poly (Glu, Tyr), 1 : 1, or poly (Glu, Ala, Tyr), 1:1:1. Proinsulin was about 100 times less active than porcine insulin in both tissues. Insulin stimulated phosphorylation of poly (Glu, Tyr), 4:1 in rat brain and liver receptors 1.7 and 5.0 times above basal respectively using similar conditions (Table 1). Autophosphorylation o f t subunit WGA purified liver and brain receptors demonstrated insulin stimulated autophosphorylation of the fl subunit (Figs. 7, 8). Basal incorporation of 32p w a s high in liver receptors, and insulin stimulated autophosphorylation was less marked than with brain receptors. Anti-receptor antiserum (Bs) immunoprecipitated the fl subunit of both brain and liver. The immunoprecipitation of brain fl subunit was less using B10and almost absent with liver receptors. The apparent molecular weight of the fl subunits were 92 kDa for brain and liver (Figs. 7, 8). A phosphoprotein band of 105 kDa (pp 105) was apparent in the brain preparation only and its phosphorylation was stimulated by insulin (10-7 mol/1) (Fig. 7, lane A, B). Anti-receptor antiserum (B8 and Bt0) failed to immunoprecipitate this protein (Fig. 7, lanes D, F). In addition, anti-pp 120 antiserum was incapable of immunoprecipitating this phosphoprotein (Fig.7, lanes G-H). Discussion Specific insulin receptors are present in liver and brain of the lizard Anolis carolinesis, lzsI-insulin binding to membrane preparations from liver and brain was time, temperature and pH dependent in a manner characteristic of insulin receptors found in tissues from other species [ 7-17 ]. Specificity studies also demonstrated competition binding curves typical for insulin receptors with chicken insulin being 2-3 times more potent than porcine insulin, and with proinsulin about 100times less potent. Liver crude membranes demonstrated higher specific binding per mg protein compared to brain crude membranes, whereas brain receptors demonstrated greater apparent affinity, with a further increase in apparent affinity following WGA purification [ 25 ]. In comparison, insulin binding to rat brain and liver membranes was similar. The difference between lizard brain and liver insulin binding is not readily apparent and requires further investigation. Crosslinking of 125I-insulinto the a-subunit of lizard liver and brain receptors revealed different apparent molecular weights on SDS-PAGE. The a-subunit of brain receptors migrated with an apparent molecular weight of 120 kDa whereas that for liver was 135 kDa. This difference has been previously reported in rats when comparing brain receptors to non-neural receptors [ 19-22 ]. It has been suggested that the lower molecular weight of brain a-subunit may be due to differences in carbohydrate residues when comparing a-subunits to non-neural tissues [ 19-22 ]. Evidence for this in lizards is two-fold. Firstly, neuraminidase, an exoglycosidase that cleaves terminal sialic acid residues, did not affect the electrophoretic mobility of lizard brain asubunits whereas digestion with neuraminidase decreased the apparent MW of liver a-subunits. Similar findings have been found in the rat [ 21, 22 ]. Secondly, though both brain and liver receptors from lizard adsorbed to and were eluted from wheat germ agglutinin columns using N-acetyl-D-glucosamine, the elution pattern of the brain receptor differed from liver. Brain insulin receptors continued to elute from the columns many fractions after the major protein peak had eluted, suggesting an unusual interaction with the column. These findings suggest that the major portion of the peak protein fraction of the WGA eluate is comprised mostly of unrelated proteins. Since glycoprotein interactions with wheat germ agglutinin are generally considered to be due to terminal sialic acid residues on the glycoprotein, the lack of effect of neuraminidase may be interpreted as indicating the absence or resistance of sialic acid residues on the brain receptor subunits [22]. Alternatively, other residues may be present on brain insulin receptors. Further studies with other lectin columns may shed more light on these differences. Both brain and liver receptors from the lizard undergo autophosphorylation of the fl-subunit. This autophosphorylation was stimulated by 10-7mol/1 insulin. Thus, despite the difference in a subunits between brain and liver receptors, these studies demonstrate a coupling between insulin binding to the a subunit and insulin stimulated autophosphorylation of the fl-subunit. Further evidence for coupling of a and fl-subunits is the ability of brain and liver receptors to phosphorylate exogenously added tyrosine specific artificial substrates that were stimulated by insulin. The phosphorylation of these tyrosine containing substrates was most efficient for poly (Glu, Tyr), 4:1 and least efficient using the polymer (Glu, Tyr), 1 : 1, which is typically seen with insulin receptors [ 30 ]. Furthermore, proinsulin was 100times less potent than porcine insulin, suggesting that the phosphorylation of the artificial substrates by both brain and liver was via the insulin receptor. The relatively higher basal phosphorylation of exogenous substrates by liver receptors compared to brain receptors may be in part related to effects by other receptors, e.g. epidermal growth factor or insulin-like growth factor receptors. This may then also account for the less marked stimulation of phosphorylation by insulin seen with liver receptors compared to brain receptors. A prominent finding was the presence of an endogenous phosphoprotein with apparent molecular weight of 105 kDa in the WGA-purified brain preparations (named ppl05). Phosphorylation of this protein was stimulated by insulin. The lack of immunoprecipitation by anti-receptor antibodies suggests that it is probably not a component of the insulin receptor. Recently, investigators have described the possible presence of endogenous substrates for phosphorylation by the insulin receptor. Rees-Jones et al. [ 31 ] demonstrated the presence of an endogenous substrate of apparent molecular weight of 120kDa (pp120) in liver membranes from dexamethazone treated rats, and Sadoul et al. [ 32 ] found a protein substrate (pp 110) in rat hepatocytes and rabbit adipocytes. Using the antibody directed towards pp120 [ 31 ], we failed to immunoprecipitate the lizard brain phosphoprotein (ppl05) [ 31 ]. This phosphoprotein may, however, be similar to that described in rat hepatocytes and rabbit adipocytes [ 32 ]. Further investigation is required to substantiate its identity and function as a possible endogenous substrate for the insulin receptor. Antireceptor antibodies did, however, immunoprecipitate the 92kDa/3 subunit of lizard brain and liver. This suggests that lizard insulin receptors probably share some antigenic determinants with both h u m a n and rat insulin receptors [ 23 ]. In conclusion, brain and liver from lizards contain specific insulin receptors which demonstrate the presence of both a a n d / 3 subunits. The a subunit of the brain has an apparent molecular weight smaller than that of the liver. Despite these differences, coupling between the a and/3 subunits is apparent as demonstrated by autophosphorylation of the/3 subunits as well as by tyrosine specific phosphorylation of artificial substrates. Thus, the presence of functional insulin receptors in liver and brain is present in the reptile kingdom. The difference between brain and non-neural receptors is also evolutionarily highly conserved at least as early as reptiles, which are thought to have arisen more than 300,000,000years before mammals [ 33 ]. Although the function of insulin in the nervous system is not completely understood, the presence of specific insulin receptors in the brain strongly suggest that insulin may well have a function in the nervous system. Whether the structural differences between the brain insulin receptors and non-neural insulin receptors are related to a particular function needs to be studied. Acknowledgements.We are grateful to Drs. J. Roth, S.Taylor, Y.Zick, N.Perrotti, J. Simon and C. Hart for helpful suggestions and V.Katz for expert secretarial assistance. We wish to acknowledge the grant for Dr. J. Shemer provided by an award to Dr. J. Roth from the Lita Annenberg Hazen fund. 1. Kasuga M , Hedo JA , Yamada KC , Kahn CR ( 1982 ) The structure of insulin receptor and its subunits . J Biol Chem 257 : 10392 - 10400 2. Ullrich A , BellJR, Chert EY , Herrera R , Petruzzelli LM , DullTJ, Gray& Coussens L, LiaoYC, TsubokawaM, Mason A , Seeburg PH , GrunfeldC, Rosen OM , RamachandranJ ( 1985 ) Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes . Nature 313 : 756 - 761 3. Ebina Y , Ellis L , Jarnagin K , Edery M , Graf L , Clauser E , Ou J-H , Maslarz F , Kan YW , Goldfine ID , Roth RA , Rutter WJ ( 1985 ) The human insulinreceptor eDNA: the structural basis for hormoneactivated transmembranesignalling . Cell 40 : 747 - 758 4. Van Obberghen E ( 1984 ) The insulin receptor: its structure and function . Biochem Pharmacology 33 : 889 - 896 5. Kasuga M , Karlsson FA , Kahn CR ( 1982 ) Insulin stimulates the phosphorylation of the 95,000 dalton subunit of its own receptor . Science 215 : 185 - 187 6. Zick Y , Rees-Jones RW , Roth J ( 1982 ) Insulin-inducedphosphorylation of the insulinreceptor: a very early event at the target cell . Proceedings of the 1lth Congress of the I. D. F. Excerpta Medica , Amsterdam Oxford Princeton, pp 161 - 170 7. Gavin III JR, Gorden P , Roth J , Archer A , Buell DN ( 1973 ) Characteristics of the human lymphocyte insulinreceptor . J Biol Chem 248 : 2202 - 2207 8. OlefskyJM, Jen P , Reaven GM ( 1974 ) Insulin binding to isolated human adipocytes . Diabetes 23 : 565 - 571 9. Freychet P , Roth J , Neville Jr DM ( 1971 ) Insulin receptors in the liver specific binding of [12sI]insulinto the plasma membrane and its relation to insulin bioactivity . Proc Natl Acad Sci USA 68 : 1833 - 1837 10. Gammeltoft S , GliemannJ ( 1973 ) Bindingand degradation of a25Ilabeled insulin by isolated rat fat cells . Biochem Biophys Aeta 320 : 16 - 32 11. Havrankova J , Roth J , Brownstein M ( 1979 ) Concentrations of insulin and of insulinreceptors in the brain are independent of peripheral insulinlevels: Studies of obese and streptozotocin-treated rodents . J Clin Invest 64 : 636 - 642 12. GinsbergBH, Kahn CR , Roth J ( 1977 ) The insulinreceptor of the turkey erythrocyte:similarity to mammalian receptors . Endocrinology 100 : 82 - 90 13. SimonJ, Freychet P , Rosselin G ( 1977 ) A study of insulinbinding sites in the chicken tissues . Diabetologia 13 : 219 - 228 14. Posner BI , Kelly PA , Shin RPC , FiesenHG ( 1974 ) Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization . Endocrinology 95 : 521 - 531 15. Muggeo M , Van ObberghenE , KahnCR, RothJ, GinsbergBH, De Meyts P, EmdinSO, FalkmerS ( 1979 ) The insulin receptor and insulin of the atlantic hagfish. Extraordinary conservation of binding specificity and negative cooperativity in the most primitive vertebrate . Diabetes 28 : 175 - 181 16. MuggeoM, GinsbergBH, RothJ, Neville DM , De MeytsP , Kahn CR ( 1979 ) The insulinreceptor in vertebrates is functionally more conserved during evolution than insulinitself . Endocrinology 104 : 1393 - 1402 17. HavrankovaJ, Roth J , Brownstein M ( 1979 ) Insulin receptors are widely distributed in the central nervous system of the rat . Nature 272 : 827 - 829 18. Woods SC , Lottes EC , McKay LD , Porte Jr D ( 1979 ) Chronic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons . Nature 282 : 503 - 505 19. Ciaraldi T , Robbins R , Leidy JW , Thamm P , Berhanu P ( 1985 ) Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells . Endocrinology 116 : 2179 - 2185 20. Yip CC , Moule ML , Yeung CWT ( 1980 ) Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling . Biochem Biophys Res Comm 96 : 1671 - 1678 21. Heidenreich KA , Zahniser NR , Berhanu P , Brandenburg D , Olefsky JM ( 1983 ) Structural differences between insulin receptors in the brain and peripheral target tissues . J Biol Chem 258 : 8527 - 8530 22. Hendricks SA , Agardh C-D , Taylor SI , Roth J ( 1984 ) Unique features of the insulin receptor in rat brain . J Neurochem 43 : 1302 - 1309 23. Rees-J0nes R , Hendricks SA , Quarum M , Roth J ( 1984 ) The insulin receptor of rat brain is coupled to tyrosine kinase activity . J Biol Chem 259 : 3470 - 3474 24. Lowry OH , Rosebrough NJ , Farr AL , Randall RJ ( 1951 ) Protein measurement with the folin phenol reagent . J Biol Chem 193 : 265 - 268 25. Hedo JA , Harrison LC , Roth J ( 1981 ) Binding of insulinreceptors to lectins: evidence for common carbohydrate determinants on several membrane receptors . Biochem 20 : 3385 - 3390 26. UdenfriendS, Stein S , Bohlen P , Dairman W ( 1972 ) Fluorescamine: A reagent for assay of amino acids, peptides, proteins and primary amines in the picomole range . Science 178 : 871 - 872 27. Harrison LC , ItinA ( 1980 ) Purification of the insulin receptor from human placenta by chromatography on immobilized wheat germ lectin and receptor antibody . J Biol Chem 255 : 12066 - 12072 28. Taylor SI , Samuels B , Roth J , Kasuga M , Hedo JA , Gorden P , Brasel DE , Pokurqa T , Engel RR ( 1982 ) Decreased insulinbinding in cultured lymphocytes from two patients with extreme insulin resistance . J Clin Endocrin01 Metab 54 : 919 - 930 29. Zick Y , Kasuga M , Kahn CR , Roth J ( 1983 ) Characterization of insulin-mediatedphosphorylation of the insulinreceptor in a cellfree system . J Biol Chem 256 : 75 - 80 30. ZickY, GrunbergerG, Rees-JonesRW, ComiRJ ( 1985 ) Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases . Eur J Biochem 148 : 177 - 182 31. Rees-Jones RW , Taylor SI ( 1985 ) An endogenous substrate for the insulin receptor-associated tyrosine kinase . J Biol Chem 260 : 4461 - 4467 32. Sadoul JL , Peyron JF , Ballotti R , Debant A , FehlmannM, Van Obberghen E ( 1985 ) Identification of a cellular 110,000-Da protein substrate for the insulin-receptor kinase . Biochem J 227 : 887 - 892 33. MacLaughlinPJ, DayhoffMD ( 1969 ) Evolution of species and proteins: a time scale . In: DayhoffMO (ed) Atlas of protein sequence and structure, Vol4 . National Biomedical Research Foundation, S.S. Md , p39 Received: 2 December 1985 and in revised form: 28 February 1986 Dr. Derek LeRoith Diabetes Branch , NIADDK Building 10 , Room 8S-243 National Institutes of Health 9000 Rockville Pike Bethesda, MD 20892 USA


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J. Shemer, J. C. Penhos, Derek LeRoith. Insulin receptors in lizard brain and liver: structural and functional studies of α and β subunits demonstrate evolutionary conservation, Diabetologia, 1986, 321-329, DOI: 10.1007/BF00452070