Receptors for insulin and growth hormone on lymphoid cells

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[53] R e c e p t o r s for 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 L y m p h o i d Cells

By

M A X I N E A. LESNIAK, JOSE A. H E D O , GEORGE GRUNBERGER, B E R N I C E M A R C U S - S A M U E L S , JESSE R O T H , and PHILLIP G O R D E N

Introduction Radioreceptor assay (RRA) has been successfully used to measure directly the binding of peptide hormones to their cell surface receptors. 1 For insulin and human growth hormone cultured human lymphocytes have provided a valuable model system. Although lymphocytes are not usually considered target cells for these hormones, they have been used for this purpose because of ease of maintenance in culture and reproducibility of results. Of the several cultured human lymphoblastoid cell lines tested the IM-9 cell line is widely used. In this chapter we will describe the techniques in which the cultured human lymphocyte has been used not only to measure insulin and human growth hormone in radioreceptor assays but also to define properties of their respective receptors. These include (1) the binding characteristics of the insulin and growth hormone receptor, (2) the transformation of freshly isolated lymphocytes from blood to permanent cultured lines by Epstein-Barr virus (EBV) transformation, (3) the structural features of both the insulin and growth hormone receptor, and (4) the ability of the insulin receptor to serve as a tyrosinespecific protein kinase. Binding Studies for Insulin and Human Growth Hormone

Cultured IM-9 Lymphocytes Reagents Cell line: IM-9 lymphoblastoid cells can be purchased from the American Type Culture Collection (Rockville, MD). The IM-9 lymphocytes are grown as a suspension culture in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) in flasks in a 37° tissue culture incubator. The cells are maintained in culture by twice weekly resuspending them at a 1 : 5 dilution with fresh medium (feed1 p. Gorden and B. D. Weintraub, in "Williams' Textbook of Endocrinology" (J. D. Wilson and D. W. Foster, eds.), p. 133. Saunders, Philadelphia, Pennsylvania, 1985.

METHODS IN ENZYMOLOGY,VOL. 150

Copyright© 1987by AcademicPress, Inc, All rightsof reproductionin any form reserved.

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ing). Cells are used for binding at 48 to 72 hr after feeding, i.e., when in late exponential or early stationary phase of growth, the density of cells is - 106 cells/ml Binding buffer: HEPES (100 mM) with NaC1 (120 mM), MgSO4 (1.2 mM), KCI (2.5 mM), sodium acetate (15 mM), glucose (I0 mM), EDTA (1 mM), and bovine serum albumin (BSA) at 10 mg/ml. (The buffer is adjusted to pH 7.2 for the human growth hormone (hGH) assay and to pH 7.8 for the insulin assay) Labeled peptides: 125I-Labeled insulin: porcine insulin radioreceptor grade can be purchased (New England Nuclear, Boston, MA, or Amersham, Chicago, IL) or prepared in the laboratory 2 125I-Labeled hGH: Prepared in the laboratory. 2 (Note: Labeled peptides that are designated for use in RIA are not suitable for RRA) Unlabeled peptides: Pork insulin is purchased from Elanco, Inc. (Indianapolis, IN). A stock solution, 1 mg/ml in 0.01 N HCI, is very stable and can be stored for at least 1 year or longer at 5°. hGH can be purchased (Genetech, San Francisco, CA) or obtained as a gift from the National Hormone and Pituitary Agency (Baltimore, MD). A stock solution, 1 mg/ml in 0.1 M NaHCO3 adjusted to pH 9.0, has limited stability in solution and is stored at - 2 0 ° (Note: All above solutions are prepared in plasticware.)

Procedure. Table I shows the composition of the incubation mixtures for the assay of hGH and insulin. Methods for the determination of the binding parameters have been described in detail elsewhere. 3,4 For the assay of complex solutions (plasma, urine, tissue culture medium, cell homogenates, crude extracts, etc.) in which the hormone represents only a small fraction of the material or is in the presence of interfering substances (proteases, salts, etc.) a gel filtration step is required prior to testing in RRA. For the insulin RRA there are at least three advantages to these procedures: (1) interfering substances may be removed; (2) proinsulin-related materials can be separated from insulin as well as the insulinlike growth factors (IGF-I and -II); (3) lyophilization can be used as a concentrating step. For special caveats related to the hGH RRA see the section on regulatory RRA. After the incubation, replicate 200-/.d aliquots are removed from the tubes and transferred to individual microfuge tubes (400/~1) containing 150/zl of chilled (4°) binding buffer. Before removing each aliquot, care is taken to be certain that the cells are homogeneously suspended. [MicroJ. Roth, this series, Vol. 37, p. 223. 3 j. Roth, this series, Vol. 37, p. 66. 4 p. De Meyts, in "Methods in Receptor Research" (M. Bleeher, ed.), p. 301. Dekker, New York, 1976.

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fuge tubes (1.5 ml capacity) can also be used for RRA when transfer of aliquot is not desired--see Regulatory RRA below.] The tubes are centrifuged for 1 min at room temperature in a microfuge. The supernatant is aspirated and discarded. Cold binding buffer (200/zl) (to dilute residual supernatant as well as to wash cell pellet) is added to each microfuge tube which is spun in the microfuge briefly. The supernatant is then aspirated, the tubes are inverted and, if necessary, the last traces of buffer are removed from around the cell pellet by capillary action with a fine-tipped pipet without disturbing the pellet. The tip of the tube containing the cell pellet is excised and the radioactivity is determined. This represents the bound radioactivity. Free radioactivity is found by subtracting bound from total. Total radioactivity is usually measured by pooling the incubation medium still remaining in the incubated tubes and determining the radioactivity in a 200-/xl aliquot; again care is taken so that the cells are homogeneously resuspended before removing the aliquot. Bound radioactivity is usually related to free (B/F ratio)or total (B/T, or B/T x 100 = % bound) radioactivity. Since it is assumed that labeled and unlabeled hormone behave identically, the specific activity of hormone is identical in both phases (cell-bound and supernatant) and the fraction of radioactive hormone in each phase reflects the fraction in that phase of the total hormone. To allow interassay comparison, it is important to refer the data to the actual cell concentration in each experiment; this is also important for the determination of parameters like receptor concentration/cells. The easiest method to count cells is the electronic counter, which performs a single count with a counting error of 1-2% (see this series, Vol. 108 [6]). However, the most practical method in most laboratories is by hemocytometer count. Under the best conditions, this method is subject to no less than 10% variation. Cell viability should be measured by standard procedures (see this series, Vol. 108 [6]). Cell viability should be 90% or greater. When cell viability is lower, specific binding is decreased, nonspecific binding is increased, and the reliability of the assay system is impaired. Comments. (1) The IM-9 cell line was first established in culture in 1971 from a female patient with multiple myeloma. 5 (2) Although we routinely test each lot of FCS, we have found that IM-9 cells can grow in almost any lot of FCS. However, it has been observed that when IM-9 cells are grown in continuous suspension for about 12 months, (from the time they are first removed from the frozen suspension) there is a progressive loss in hGH receptors but no apparent loss in insulin receptors. 5 j. L. Fahey, D. N. Buell, and H. C. Sox, Ann. N . Y. Acad. Sci. 190, 221 (1971).

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(3) The addition of antibiotics to growth medium does not noticeably change the binding properties of these cells.

Epstein-Barr Virus-Transformed Human L ymphocytes The insulin receptor can be studied in human lymphocytes transformed by Epstein-Barr virus. The transformed cells are a valuable asset for the study of insulin receptors both in normal controls and patients,Sa because each individual's lymphocytes can be grown indefinitely and in large quantity. 6,7

Reagents Solutions Plasmagel (HTI Corp, Buffalo, NY) Phosphate-buffered saline (pH 7.4) (PBS) RPMI-1640 tissue culture medium FCS (heat-inactivated at 56° for 30 min) Trypan blue solution (0.4% in normal saline) Polybrene (Sigma Chemical Company, St. Louis, MO) ACK lysing buffer (ammonium chloride, 8.29 g/liter; potassium bicarbonate, 1.0 g/liter; disodium EDTA, 37 mg/liter, pH 7.4) Epstein-Barr virus: Approximately 2.5 × 108 transforming U/ml is prepared from medium of marmoset B-95-8 cells. [This virus can be supplied frozen by Showa University Research Institute for Biomedicine in Florida, St. Petersburg, FL, or prepared by the investigator in the following manner. The medium in which the virus-infected B-95-8 cells (American Type Culture Collection, Rockville, MD) has been growing is clarified by centrifugation at 60 g for 10 min. The supernatant is filtered through a 0.45-/.~m Millipore filter. The virus, under sterile conditions, is pelleted by centrifugation at 10,000 g for 2 hr. This pellet is resuspended in growth medium RPMI-1640 with 10% FCS. The final volume for suspension is 1/300 of starting volume.]7a To eliminate multiple freeze-thaw steps of virus, the virus is aliquoted into 50-/zl volumes; each 50-/~1 aliquot is diluted to 3-5 ml before use. The preparations of virus are stable for at least 6 months at -70 °. This virus is a potential biohazard. Appropriate care must be taken when using and disposing of wastes 5a G. Armstrong, personal communication. S. I. Taylor, L. H. Underhill, J. A. Hedo, J. Roth, M. Serrano Rios, and R. M. Blizzard, J. Clin. Endocrinol. Metab. 56, 856 (1983) 7 S. I. Taylor and B. Marcus-Samuels, in "Insulin Receptors" (R. De Pirro and R. Lauro, eds.), p. 111. Acta Medica, Rome, 1985. 7a M. Nonoyama, personal communication.

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Sterile 15-ml screw-top conical plastic tissue culture tubes Sterile 12 x 75 mm clear plastic tissue culture tubes with caps Sterile 25-cm 2 tissue culture flasks Preservative-free heparin Procedure

1. Once blood (15-30 ml) has been drawn from the patient using sterile techniques, the following steps should be performed under sterile conditions. Preservative-free heparin (25 U/ml blood) is added to the blood. The blood should be processed as soon as possible but may be stored on ice for up to 24 hr. 2. Warm the blood to room temperature if it has been chilled. Transfer the blood to sterile 15-ml screw-top tubes and add the Plasmagel (0.5-1.0 ml/ml of blood). Place the tubes in a 37° tissue culture incubator at a 45 ° angle for 15 min, then in a vertical position for an additional 10 min. Transfer the supernatant to 15-ml screw-top conical tubes. 3. Separate the cells from the plasma by centrifugation at 400 g for 10 min at room temperature (the cell pellet will appear red because of the erythrocyte contamination). Discard the supernatant and resuspend the cells in l0 ml of PBS and repeat the centrifugation. Discard the supernatant and resuspend the cells in 10 ml of ice-cold ACK lysing buffer and store on ice for 10 min. (The function of the ACK lysing buffer is to lyse the erythrocytes.) Centrifuge the cells as specified above. Wash the cells two to three times with I0 ml of PBS and centrifuge again as specified above. 4. Resuspend the cells at a density of 5 x 10 6 cells/ml in RPMI-1640 medium containing 10% FCS [plus Polybrene (2 mg/ml--optional)]. (The yield ofleukocytes is usually approximately 2-10 × l06 cells/ml of blood.) Place 1-ml aliquots of suspended cells into each of 12 loosely capped, sterile, 12 × 75 mm tubes and incubate overnight at 37° in a tissue culture incubator to allow cells to adapt to the growth medium environment. 5. The following day prepare the Epstein-Barr virus by thawing it quickly in a water bath at 37°. The virus is then diluted in RPMI-1640 plus 10% FCS at 4 °. Keep the dilutions of virus on ice and use them as soon as possible. (It is prudent to wear gloves while handling the virus. Dispose of wastes as if they were a biohazard.) Use the virus at a concentration of approximately 2 x l05 transforming U/ml. However, a high success rate has been observed with concentrations 10- to 100-fold lower. 6. Remove the tubes from the incubator, taking care not to disturb the cells. With a pipet aspirate and discard the medium. Add 0.2-ml aliquots of the diluted Epstein-Barr virus to each of the tubes. Add 0.2-ml aliquots of RPMI-1640 to each of two tubes which will serve as controls (i.e., no viral infection). Incubate the tubes for 2 hr at 37°.

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7. After 2 hr, to each tube add 2 ml RPMI-1640 medium plus 10% FCS at room temperature and centrifuge at 400 g for I0 min at room temperature. Aspirate and discard the medium. Add 1 ml of fresh RPMI-164010% FCS and resuspend the cells by tapping the tubes. Return the tubes to the tissue culture incubator, keeping them loosely capped. Fresh growth medium must be added to the cells every 3-4 days. This is done by removing approximately 0.5 ml of medium and replacing it with an equivalent volume of fresh medium. After 3-6 weeks, the medium should have visible clumps of transformed cells. At this point, it is necessary to transfer the cells to 25-cm 2 tissue culture flasks. The contents of three to four tubes are added to one flask with an equal volume of fresh tissue culture medium. Once the cells are growing they can be fed by adding fresh medium every 3-5 days, depending on the growth rate. It is preferable to separate the cells from the growth medium by centrifugation (250 g, 10 min, room temperature) and to resuspend the cells in fresh medium. However, it is also possible to simply dilute the cells 2- to 4-fold with fresh medium. Cells may be stored frozen in 10% glycerol or 7.5% dimethyl sulfoxide under usual procedures (see also this series, Vol. 108 [36]). When the cells have reached the growing stage they are ready to be used in the insulin RRA as described in Table I. Figure 1 shows insulin

TABLE I COMPOSITION OF INCUBATION MIXTURES

Growth hormone assay a Binding buffer (pH 7.2) (to give volume of 500/~1) txsI-Labeled hGH Unlabeled hGH (or unknown) Cells (cultured lymphocytes IM-9) Insulinb Binding buffer (pH 7.8) (to give volume of 500/~1) t25I-Labeled insulin Unlabeled insulin (or unknown) Cells (cultured lymphocytes IM-9)

Volume (/zl)

Final concentration

0-50 50 0-50 400

0.50 ng/ml 0-1.0 gg/ml 20 x 106/ml

0-50 50 0-50 400

0.2 ng/ml 0-10 ~g/ml 2.5 x 106/mi

a Incubation conditions: 90 min in 30° waterbath. Intermittent Shaking of tubes to disperse cells during incubation time is suggested. (Note: When cells are aliquoted to assay tubes (12 × 75 cm plastic or 1.5-ml microfuge tubes) one needs to disperse cells by manual shaking (not vortex shaking, which can disrupt cells) between each addition. Addition of cells to tubes is the last step, and initiates the time of incubation.) Incubation conditions: 2 hr in a 15° waterbath. (See footnote a regarding shaking.)

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35

30

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FIG. 1. Competition curves for 125I-labeledinsulin to Epstein-Barr virus-transformed cultured lymphocytes. Shaded area denotes the range of binding of labeled insulin to cultured lymphocytes from normal subjects and insulin-dependent diabetic patients; @--@ denotes binding to cultured lymphocytes from a patient with Rabson-Mendenhall syndrome .6

RRA data obtained using EBV-transformed lymphocytes. Neither hGH nor insulin-like growth factor receptors have been detected in virus-transformed lymphocytes. Comments. (1) In this method, Plasmagel has been used to separate the leukocyte fraction from the blood. It is possible to substitute a FicollHypaque centrifugation technique to isolate the mononuclear leukocyte fraction. We prefer the Plasmagel technique because it is simple and reliable. (2) It is difficult to establish the optimum time for the transfer of the cells from test tubes to tissue culture flasks. The growth of cells should be checked several times a week using an inverted microscope. When, under low power, there are many obvious visible clumps of viable cells, it is time to transfer the cells to the flasks. If there is any doubt, it is safer to refeed the cells and postpone the transfer. If the cells are transferred too early, they may not survive. (3) Our present success rate in

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establishing transformed cell lines is approximately 80-90%. (4) The disadvantage of using cultured lymphocytes is that they can only be used to study receptor function per se; up to now we have been unable to demonstrate typical biological responses to insulin on these cells. (5) Other cultured human lymphocytes (both B and T cell lines) have been tested for their ability to bind insulin and human growth hormone. Cell lines of B cell type display a wide spectrum of binding for both hormones while T cell types have low binding for insulin and no binding for growth hormone. 4 Regulation Plus Competition Assay The binding of a hormone to specific receptors is a rapid, reversible, and saturable process. Several studies have indicated that the concentration of receptors on cells can be regulated by the level of hormone to which the cells are being exposed; the terms "down regulation" and "desensitization" have been used to describe this effect. (This phenomenon was first demonstrated for insulin using IM-9 lymphocytes by Gavin e t al. 8 and later for hGH in the same cells by Lesniak and Roth. 9) Thus, unlabeled hormone can reduce the binding of labeled hormone in two ways: first, by competing with the labeled hormone for occupancy of a fixed number of binding sites (competition RIA is a prototype for this kind of assay) and, second, by interacting with the cells to produce a decrease in the total number of receptors, thereby reducing the total number of sites available for occupancy by both unlabeled and labeled hormone (regulation). Thus, regulation by receptor-mediated endocytosis, or possibly other mechanisms that recruit receptors, may be a more sensitive function to measure hormone concentration than competition alone. Experimentally, this regulation effect has been demonstrated for hGH. Under conditions that induce endocytosis of the hGH receptor, a concentration of hGH as low as 10-10 M will cause a decrease in binding of the labeled hormone, whereas in competition studies at least an order of magnitude greater hormone concentration is necessary to cause a decrease in binding. We have taken advantage of these two properties, regulation and competition, to broaden the scope as well as to increase the sensitivity of the radioreceptor assay for hGH. (Theoretical discussion of this regulation plus competition assay has been previously published. 1°) There are other mechanisms which may act in concert with 8 j. R. Gavin, III, J. Roth, and D. M. Neville, Jr., Proc. Natl. Acad. Sci. U.S.A. 71, 84 (1974). 9 M. A. Lesniak and J. Roth, J. Biol. Chem. 251, 3720 (1976). 10 R. C. Eastman, M. A. Lesniak, J. Roth, P. De Meyts, and P. Gorden, J. Clin. Endocrinol. Metab. 49, 262 (1979).

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competition to increase the sensitivity of the RRA. For instance, it is known that bound ~25I-labeled insulin is reversibly dissociated from cells upon dilution and the addition of unlabeled insulin will increase the rate of dissociation over dilution alone. This phenomenon, known as "negative cooperativity," increases the sensitivity of the insulin RRA.t° Measurement of hormone with the insulin RRA, however, requires no changes in the assay protocol. The regulation plus competition assay described here is specific for hGH.

Procedure and Reagents Reagents are as described in RRA section (see above). Cells at 1-4 x 107/ml are diluted in 0.40 ml binding buffer and either unlabeled hGH (0104 ng/ml in 0.05 ml buffer) for the standard curve or unknown sample (0.05 ml) is added. After 4.5 hr of incubation at 30°, ~25I-labeled hGH (250 pg in 0.05 ml buffer) is added to give a final volume of 0.5 ml, and the incubation is continued for an additional 1.5 hr, i.e., a total of 6 hr. At the end of the incubation, the separation of the bound and free hormone is performed as previously described for the competition assay. Figure 2 shows the data obtained using this assay procedure.

0r r z~" 0 U

90 80

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FIG. 2. Comparison of two types of radioreceptor assays. IM-9 lymphocytes were incubated at 30° in the absence ( I - - O , competition only) or presence ( O - - O , regulation plus competition) of unlabeled hormone for 4.5 hr, followed (for both) by 1.5 hr in the presence of labeled hormone ( O - - O ) or unlabeled and labeled hormones (Q--O). Total binding (15%) has been normalized to 100% and is plotted as a function of the total concentration of unlabeled hormone; nonspecific binding has not been subtracted.I°

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Adaptation of Procedure for Samples Obtained from Biological Fluids As previously mentioned, serum or plasma may nonspecifically interfere in the competition assay or the regulation plus competition assay. In an attempt to avoid the gel filtration or some other purification step Gavin et ai. proposed a modification to the regulation plus competition assay. H In this modification, 20% hGH-free serum (i.e., serum obtained from individuals with hGH deficiency) is added to the standard curve; the patient's serum at a dilution up to 20% can be added directly. At the end of a 90-min incubation the cells are washed twice in binding buffer and labeled hormone is added as described above. In this modification the serum blank and the wash step are used to minimize nonspecific interference by serum. While these modifications have advantages, especially for measuring hormone concentration in small serum samples, they do not completely eliminate nonspecific interference. Thus, we purify 5 ml of serum or plasma on a Sephadex G-100 column (I .5 × 90 cm). Components of hGH (22,000 Da, 20,000-Da variant, higher molecular weight oligomers) are, under these conditions, at a concentration and purity adequate to be measured in RRA.

Comment (I) The hGH assays using IM-9 lymphocytes are specific for human GH. Nonprimate growth hormone except at very high concentrations does not react with the human GH receptor. (2) The competition hGHRRA is poorly sensitive to physiological concentrations of peptide components. The hGH-RRA (and insulin RRA) is at least an order of magnitude less sensitive than the RIA. The enhanced sensitivity achieved by regulation plus competition assays for peptides such as hGH is of considerable value. (3) Previously, we had described the isolation of components from serum by gel filtration techniques using volatile buffers for elution and lyophilization as a concentrating step. While these steps are still suitable for separating and measuring insulin components, hGH plasma components may be unstable under these conditions; thus somewhat larger volumes of serum may be necessary for the gel filtration of hGH. Receptor Labeling Techniques for Insulin and Growth Hormone Receptors Hormone receptors such as those for insulin and growth hormone are integral membrane proteins of very low abundance. In order to study their a j. R. Gavin III, B. Trivedi, and W. H. Danghaday, J. Clin. Endocrinol. Metab. 55, 133 (1982).

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molecular properties, a commonly used experimental approach consists of the introduction of a radioactive label in the receptor and its subsequent analysis on sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. The techniques available to radioactively label hormone receptors can be classified into two major groups: affinity and general labeling methods. In affinity-labeling methods, the radioactive biological ligand is allowed to bind to the receptor and then a covalent bond between the ligand and the receptor is created with the use of either homobifunctional or photoreactive heterobifunctional reagents. The major advantage of the affinity-labeling methods is their specificity since in theory only the receptor is labeled. On the other hand, one of their disadvantages is that in the case of oligomeric receptors with dissimilar subunits, only the binding subunit is detected. In addition, the introduction of a covalent bond between the ligand and the receptor usually alters the physiological fate of the receptor and makes these methods inappropriate for the study of the turnover and life cycle of the receptor. General labeling methods consist of the introduction of radioactivity in many, if not most, cellular proteins, among which the receptor usually represents a very small fraction. This type of labeling is accomplished by the biosynthetic incorporation of labeled amino acids or sugars, or via chemical methods for the iodination or tritiation of exposed cell surface proteins or glycoproteins. Since large pools of cellular proteins are labeled simultaneously, it is necessary to select and isolate the receptor subsequently with the use of immunological probes such as anti-receptor antibodies. These methods have greater scope and applicability than affinity-labeling techniques since they allow the study not only of the structural features of the receptor but also of its biosynthesis and turnover. The major limitation is the availability of anti-receptor antibodies in some cases. Obviously the specificity and sensitivity of these methods are always dependent on the properties of the immunological probes used.

Affinity Labeling with Homobifunctional Reagents Insulin Receptor. Theory: Bifunctional reagents carry two identical reactive groups and can be used to introduce a covalent bridge between lz~I-labeled insulin and its receptor. In theory, the use of these reagents poses several problems since they can indiscriminately and extensively cross-link many membrane proteins and their subunits in addition to the ligand and the receptor. Pilch and Czech introduced in 1979 the use of disuccinimidyl suberate to cross-link 125I-labeled insulin to its recep-

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t o r . 12,13 This reagent has proved to be very useful and has been used thereafter extensively to study insulin receptors in lymphoid cells. 14-16 Disuccinimidyl suberate reacts with amino groups and has an approximate span of 11/~.

Reagents IM-9 lymphocytes at a stationary phase of growth, 108 cells Labeled, unlabeled insulin, and buffer with and without 0.1% BSA as described in reagents for RRA (see above) Disuccinimidyl suberate (Pierce Chemical Company) dissolved in dimethyl sulfoxide (10 mM stock solution) Stopping buffer, 100 mM Tris, l0 mM EDTA, pH 7.4 PBS, pH 7.4 Procedure 1. Sediment the cells by centrifugation at 600 g for 5 min and wash with PBS three times in order to remove completely the culture medium. Resupend the cells at a density of 5 x 10 7 cells/ml in lymphocyte-binding buffer with BSA (see above). 2. Incubate the cells with 125I-labeled insulin (5 ng/ml) in the absence and presence of unlabeled insulin (10/~g/ml) for 120 min at 15°. 3. Add ice-cold binding buffer without albumin (10 ml). Sediment the cells by centrifugation at 600 g for 5 min at 4°. Discard the supernatants and resuspend the cells in 1 ml BSA-free binding buffer at 4°. 4. Add disuccinimidyl suberate dissolved in dimethyl sulfoxide to give a final concentration of 50/~M. Incubate on ice for 15 rain. 5. Stop the reaction by adding 3-4 ml of 100 mM Tris-HC1 and 10 mM EDTA, pH 7.4. After further incubation for 5 min, the cells are sedimented by centrifugation and washed once with PBS. 6. At this point the cells can be solubilized and the cross-linked receptors analyzed by SDS/polyacrylamide gel electrophoresis as described below. Comments. (1) The cross-linking reagent, disuccinimidyl suberate, can be used at a concentration between 10 and 300/xM. As the concentration of the cross-linking reagent is increased, the extent of the label bound 12 p. F. Pilch and M. P. Czech, J. Biol. Chem. 254, 3375 (1979). 13 p. F. Pilch and M. P. Czech, J. Biol. Chem. 255, 1722 (1980). 14 M. Kasuga, E. Van Obberghen, K. M. Yamada, and L. C. Harrison, Diabetes 30, 354 (1981). 15 S. I. Taylor, B. Marcus-Samuels, J. Roth, M. Kasuga, J. A. Hedo, P. Gorden, D. E. Brasel, T. Pokora, and R. R. Engel, J. Clin. Endocrinol. Metab. 54, 919 (1982). ~6A. McElduff, J. A. Schroer, and S. I. Taylor, Endocrinology (Baltimore) 115, 1869 (1984),

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is increased, but the amount of very high-molecular-weight material, which is not resolved by SDS/polyacrylamide gel electrophoresis, increases as well. The optimum concentration appears to be 50-100/~M. (2) The efficiency of cross-linking is relatively low, i.e., 10-20% of the bound ligand is covalently cross-linked to the receptor as detected by gel electrophoresis under nonreducing conditions.13 Under reducing conditions only 2-10% of the label appears to be associated with the receptor. This additional decrease may be due to the fact that the cross-linking event seems to occur through lysine B29 and/or phenylalanine B1,13 whereas most of the 1251label in monoiodoinsulin preparations is in the A chain, which is cleaved from the cross-linked complex under reducing conditions. The labeling of the receptor increases as receptor occupancy is increased by using higher concentrations of radioactive hormone in the binding reaction. The concentration of 125I-labeled insulin is usually 2-20 ng/ml, depending upon cell volume used in binding reaction. (3) In addition to intact cells, membrane fractions TM or detergent-solubilized fractions 17 can be used as a source of receptors. The procedure is similar except for the conditions of the hormone-binding reaction (e.g., temperature, buffer, incubation time, etc.), which should be optimized for each preparation. (4) Buffers containing Tris, albumin, or any other free amino groups should be avoided in the cross-linking reaction. (5) This technique and all the others in this section are described for IM-9 lymphoctyes. However, they can be used with any other type of lymphoid cells provided that the number of cells is adjusted according to the abundance of insulin receptors as determined by binding studies. Growth Hormone Receptor. Theory: The same homobifunctional reagent, disuccinimidyl suberate, used in the study of the insulin receptor, has been successfully used to cross-link covalently 125I-labeled hGH to its receptor in IM-9 lymphocytes.18 Reagents and Procedure. The experimental protocol is similar to that used for 125I-labeled insulin cross-linking and described in the previous section. The following modifications are used. 125I-Labeled human growth hormone (20-40/zCi//zg) is incubated at a concentration of 10-50 ng/ml with 2.5 × 107 cells/ml in binding buffer with 0.1% (w/v) BSA, pH 7.4, for 90 min at 30° and in the absence or presence of unlabeled hormone (1/xg/ ml). After washing with ice-cold BSA-free binding buffer the cells are resuspended in 1 ml of the same buffer. Disuccinimidyl suberate is added at a final concentration of 0.1 mM and the reaction is allowed to occur for 30 min on ice. t7 j. A. Hedo and I. A. Simpson, Biochem. J. 232, 71 (1985). is K. Asakawa, G. Grunberger, A. McElduff, and P. Gorden, Endocrinology (Baltimore) 117, 631 (1985).

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Comments. In addition to disuccinimidyl suberate, another bifunctional reagent, ethylene glycol bis(succinimidyl succinate) (Pierce Chemical Company), can be used to cross-link 125I-labeled hGH to its receptor in IM-9 lymphocytes. ~9This reagent is used at a final concentration of 0.15 mM and is cleavable by incubation with 1 M hydroxylamine at pH 8.5 and 37° for 5 hr. Affinity Labeling with Heterobifunctional Photoreactive Photosensitive Reagents General Considerations. Heterobifunctional groups carry two reactive groups, one photosensitive and one conventional. In principle, they are designed to overcome the potential problems of homobifunctional reagents such as random coilisional cross-links, considerably long reaction times, and generalized cross-linking of membrane proteins. The heterobifunctional reagents allow an easier and more rapid control of the reaction because one of the functional groups can be activated when desired by photolysis. The reagent is first attached to the ligand hormone via the conventional group. After purification and radioactive labeling, the hormone is allowed to bind to the receptor preparation. Up to this point all the steps are carded out in the dark; the hormone is then covalently linked to the receptor by exposure to ultraviolet light. Several photoreactive derivatives of insulin have been successfully used to study the structure of the insulin receptor. 2°-22 Insulin Receptor. Yip et al. 2° introduced in 1978 the first photosensitive insulin derivative 4-azidobenzoylinsulin. Jacobs et al. 21 used a similar derivative, 4-azido-2-nitrophenylinsulin. In our laboratory we have used three different insulin derivatives, 23as originally prepared by D. Brandenburg and associates. 24The best labeling results were obtained with N~-B29(2-nitro-4-azidophenylacetyl)insulin. Similar derivatives with the reactive group at the A1 o r BI residues of the insulin molecule were less effective in 19j. p. Hughes, J. S. A. Simpson, and H. G. Friesen, Endocrinology (Baltimore) 112, 1980 (1983). 20 C. C. Yip, C. W. T. Yeung, and M. L. Moule, J. Biol. Chem. 253, 1743 (1978). 21 S. Jacobs, E. Hazum, Y. Schechtcr, and P. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A. 76, 4918 (1979). M. H. Wisher, M. D. Baron, R. H. Jones, P. H. Sonksen, D. J. Saunders, P. Thamm, and D. Brandenburg, Biochem. Biophys. Res. Commun. 92, 492 (1980). C.-C. Wang, J. A. Hedo, C. R. Kahn, D. T. Saunders, P. Thamm, and D. Brandenburg, Diabetes 31, 1068 (1982). u p. Thamm, D. T. Saunders, and D. Brandenburg, in "Insulin Chemistry, Structure and Function of Insulin and Related Hormones" (D. Brandenburg and A. Wollmer, eds.), p. 309. de Gruyter, Berlin, 1980.

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receptor binding and labeling. Arylnitrenes generated from arylazides by photolysis do not require a specific reactive group and have been found to react with most amino acid residues. 25

Reagents IM-9 lymphocytes at a stationary phase of growth, 10s cells N~-B29-(2-nitro-4-azidophenylacetyl)insulin iodinated with lZSIwith the chloramine-T method (100-120/.~Ci//.~g)2 Unlabeled insulin and binding buffer with BSA 0.1% (w/v) and PBS Ultraviolet radiation source (super-pressure mercury lamp, model ALH 215, Osram HBO 100 w/z, Photochemical Research Associates, Inc.)

Procedure 1. Sediment the cells by centrifugation at 600 g for 5 min and wash with PBS three times. Resuspend the cells at a density of 5 x l07 cells/ml in binding buffer. 2. Incubate the cells with photoreactive 125I-labeled insulin (20-25 ng/ ml) in the absence and presence of unlabeled insulin (10/~g/ml) for 120 min at 15° in the dark. 3. Expose the cell suspension to ultraviolet radiation for 80 sec at a distance of 30 cm from the lamp and at room temperature. 4. Wash the cells extensively (four times) with PBS supplemented with 0.1% BSA and at room temperature to remove the free and noncovalently bound insulin. 5. At this point detergent solubilization and electrophoretic analysis can be performed as described below. Comments: The photoreactive insulin derivative should be handled at all times before photolysis in the dark or in darkroom illumination (Solar Master filter protected light). About 20% of the photoinsulin is covalently linked to the receptor under these conditions. Preliminary tests should be conducted with different types of ultraviolet lamps in order to determine optimum photolysis time and distance.

Analysis of Affinity-Labeled Receptors Affinity-labeled proteins are usually analyzed by SDS/polyacrylamide gel electrophoresis. Unlike general labeling methods, affinity-labeling techniques do not require isolation of the receptor by immunoprecipitation with anti-receptor antibodies since the labeling is already selective T. H. Ji, Biochim. Biophys. Acta

559, 39 (1979).

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for the receptor. However, when the receptor preparation is whole cells, it becomes impractical to solubilize the cells directly in SDS/electrophoresis buffer because of the large amount of proteins and nucleoproteins. Instead, the cells are solubilized first in a nonionic detergent, such as Triton X-100 (1%, v/v), unsolubilized material is removed by ultracentrifugation (200,000 g for 60 min), and the labeled receptors are then precipitated from the supernatant using acetone:concentrated ammonium hydroxide (16: 1, v/v) or trichloroacetic acid (15%, w/v) at ice bath temperature. The pellets can be finally solubilized in SDS/electrophoresis buffer. Alternatively, immunoprecipitation with anti-receptor antibodies of the Triton X-100 solubilized preparation can be used as a concentration step. Anti-ligand antibodies can also be used for this purpose. 16 The analysis of the labeled receptors is performed by SDS/polyacrylamide gel electrophoresis in slab gels and discontinuous b u f f e r systems according to Laemmli. 26 The procedures for Triton X-100 solubilization, immunoprecipitation with anti-receptor antibodies, SDS/electrophoresis, and autoradiography have been described in detail in another volume of this series? 7 Affinity-labeling procedures with homobifunctional reagents or photoreactive derivatives reveal under reducing conditions the a subunit of the insulin receptor with an apparent Mr 135,000 in 7.5% acrylamide gels. Very faint labeling or none is usually found in the fl subunit of the insulin receptor Mr 95,000.14,16,23Under nonreducing conditions several high-molecular-weight components in a region of approximately Mr 300,000 are found in 5% acrylamide gels. 23 Both cross-linking with disuccinimidyl suberate and photoaffinity labeling yield very similar results. Disuccinimidyl suberate may be a more convenientprocedure over photoreactive insulins because of its wide availability and easier handling. Affinity labeling of the hGH receptor in IM-9 lymphocytes reveals an Mr 140,000 band under reducing conditions in 7.5% acrylamide gels. 18 Under nonreducing conditions and using 5% acrylamide gels an additional complex of Mr 270,000 band is also found. Apparent Mr values on SDS gels should be always considered with caution given the anomalous behavior of glycoproteins (as are insulin and growth hormone receptors) under these conditions. Furthermore, under nonreducing conditions, Mr values should be considered as approximations given the impossibility of using appropriate molecular weight markers.

26 U. K. Laemmli, Nature (London) 227, 680 (1975). 27 j. A. Hedo, and C. R. Kahn, this series, Vol. 109, p. 593.

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General Labeling M e t h o d s

Several labeling procedures have been described to study the insulin receptor in cultured IM-9 lymphocytes. Biosynthetic labeling can be achieved with a variety of radioactive sugars and amino acids. On the other hand, cell-surface labeling can be achieved with the lactoperoxidasep25I or the galactose oxidase/NaB3H4 techniques. All these techniques have been described in detail in another volume of this series 27 (see also this series, Vol. 108 [42]). However, a change in the method for biosynthetic labeling with radioactive amino acids, such as [3H]leucine or [35S]methionine, should be mentioned here. Previously it was suggested to partially purify the solubilized receptor on immobilized lectins in order to reduce the background of nonspecifically precipitated bands to an acceptable level. 27 We have found, however, that this step can be advantageously substituted by repeated treatment of the detergent extract with the adsorbent which will be used for the immunoprecipitation. The recommended procedure is now to label the cells for 5-20 min and solubilize the cells in Triton X-100 at 4° with a large excess of the suspension of formaldehyde-fixed Staphylococcus aureus cells (repeat suspension of extract with S. aureus three times). For each suspension 200/zg of S. aureus cells should be used for each milliliter of detergent extract. Biosynthetic and cell-surface labeling methods detect clearly both major subunits of the insulin receptor of apparent Mr 135,000 and 95,000. 27 They are far superior in this sense to affinity-labeling methods. In addition, biosynthetic labeling methods have also revealed the existence of a single-chain proreceptor of both subunits with an Mr 190,000 (as detected in 7.5% acrylamide gels under reducing conditions). Another component of Mr 210,000 is also detected at the cell surface and appears to represent a full carbohydrate-processed but uncleaved form of the precursor 28 (see Table II). 29'3° Biosynthetic cell-surface labeling techniques have not been used yet for the study of the hGH receptor, due to the lack of anti-receptor antibodies with the adequate affinity and capacity. As they become available, the same procedures used for the insulin receptor in lymphoid cells should be easily adapted for the study of the growth hormone receptor. 28 j. A. Hedo, C. R. Kahn, M. Hayashi, K. M. Yamada, and M. Kasuga, J. Biol. Chem. 258, 10020 (1983). 29 A. Ullrich, J. R. Bell, E. Y. Chen, R. Herrera, L. M. Petruzzelli, T. J. Dull, A. Gray, L. Coussens, Y.-C. Liao, M. Tsubokawa, A. Mason, P. H. Seeburg, C. Grunfeld, O. M. Rosen, and J. Ramachandran, Nature (London) 313, 756 (1985). 3o y . Ebina, L. Ellis, K. Jarnagin, M. Edery, L. Graf, E. Clauser, J.-H. Ou, F. Masiarz, Y. W. Kan, I. D. Goldfine, R. A. Roth, and W. J. Rutter, Cell (Cambridge, Mass. ) 40, 747 (1985).

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TABLE II GENERAL CHARACTERISTICS OF THE INSULIN RECEPTOR Insulin receptor

Characteristics

Synthesized as precursor

Mr 190,000; single-chain protein with high mannose-type chains2a Mr 135,000; protein with complex carbohydratemoiety; contains binding region; exclusivelyextracellulara 29and cysteine.richa 29.30 Mr 95,000; protein with complexcarbohydrate moiety; contains tyrosine kinase and autophosphorylation site; transmembranea 29.30

Processed to a-subunit

Processed to/3-subunit

Data from cDNA clone.

Phosphorylation Phosphorylation and dephosphorylation of enzymes have been known to provide important regulatory mechanisms in hormone action. Recently, insulin was shown to stimulate phosphorylation of its own receptor in intact lymphoid cells) 1 Subsequently insulin-stimulated autophosphorylation was also demonstrated in cell-free systems. 32

Autophosphorylation of the Insulin Receptor in Intact Cells Insulin-stimulated receptor autophosphorylation was originally described in human IM-9 lymphocytes, 31 but these methods have been utilized with various freshly isolated and cultured cell lines. Insulin, at concentrations of 1-1000 nM, stimulates phosphorylation of the fl subunit of its receptor (Mr ~ 95,000) in every system studied thus far. This effect is rapid, occurring within 1 rain at 37°. Appearance of phosphotyrosine appears to be the initial event.

Reagents Cells RPMI-1640 medium (standard and phosphate free). Phosphate-free RPMI-1640 is a special order prepared by Grand Island Biological Company (Grand Island, NY) or Biofluids (Rockville, MD) FCS Carrier-free ortho[3ZP]phosphate (New England Nuclear, Boston, MA) Insulin (see above) 31 M. Kasuga, F. A. Karlsson, and C. R. Kahn, Science 215, 185 (1982). 32 M. Kasuga, Y. Zick, D. L. Blithe, M. Crettaz, and C. R. Kahn, Nature (London) 298, 667 (1982).

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"Stopping" solution: 50 mM HEPES, 4 mM EDTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 2 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride (Sigma), 1/~g/ml aprotinin (Sigma), pH 7.4 Triton X-100 (Du Pont/New England Nuclear, Boston, MA) N-Acetyl-~glucosamine Wheat germ agglutinin coupled to agarose (Miles-Yeda, Vector) Anti-insulin receptor antibodymobtained from patients with type B extreme insulin resistance and Acanthosis nigricans. (These antibodies can be used directly at 1 : 50 or greater dilution; IgG can be prepared but is not usually necessary) Protein A (Pansorbin, Calbiochem) "Sample" buffer: 0.5 M Tris, 10% (v/v) SDS, 50% (v/v) glycerol, 3.65 M mercaptoethanol, 0.04% (v/v) bromphenol blue, pH 7.6 Supplies for SDS/polyacrylamide gel electrophoresis Kodak X-Omat film Procedure. Cells are grown in RPMI-1640 medium plus 10% FCS; cells grown in 1-1.5 liters are used for intact cell phosphorylation studies. Cells are centrifuged (600 g, 5 min, 22 °) and washed twice with phosphatefree, serum-free RPMI-1640. Cells are resuspended in phosphate-free, serum-free RPMI-1640 medium (5 ml for each experimental condition) and are labeled with carrier-free ortho[32p]phosphate (0.5 mCi/ml of medium) for 2 hr at 37°. Five-milliliter aliquots are transferred into prewarmed (37 °) 50-ml plastic test tubes and incubated without or with insulin (typically 10-100 nM) or other ligands at 37° for 1-10 min. The reaction is stopped by pouring 20 ml of ice-cold "stopping" solution into the incubation mixture and spinning the test tubes immediately (600 g, 5 min, 4°). Supernatants are discarded and pellets solubilized with 5 ml of "stopping" solution (with I0 mM sodium fluoride) containing 1.0% Triton X-100, for 30 min with constant rotation at 4°. After centrifugation (200,000 g, 60 rain, 4 °) the supernatants (solubilized material) are combined and then applied onto wheat germ agglutinin-agarose columns (0.5 ml). The columns are washed with 50-75 vol of stopping solution (with 10 mM sodium fluoride) containing 0.1% Triton X-100. The insulin receptor-enriched material (glycoproteins) is eluted with two 0.5- to 1-ml aliquots of 0.3 M N-acetyl-D-glucosamine. The insulin-receptor eluates are incubated with anti-insulin receptor antibodies (usual final titer I : 100 to 1 : 250 human serum antibodies) or with normal serum in 1.5-ml microfuge tubes for 16 hr at 4°. Immune complexes are precipitated with protein A (Pansorbin), at ~ 0.1 vol of the incubation mixture (30 min, 4° with constant rotation). Immunoprecipitates are washed twice with 0.75 ml solution of 50 mM HEPES, 1% Triton

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X-100, and 0.1% SDS and once with 50 mM HEPES and 1% Triton X-100. Electrophoresis "sample buffer" (1 × concentration; 0.1 ml) is added and pellets are resuspended. The samples are boiled (100°, 5 min) to complete the reduction of eluted proteins. Phosphoproteins are analyzed by SDSpolyacrylamide electrophoresis (Laemmli method) 26 and autoradiography. Comments. (1) This procedure can be used with various freshly isolated cells or cultured cell lines. We have used the method in studies of patients with insulin resistance (EBV-transformed cultured B lymphocytes) and in studies of more basic biochemical mechanisms with hepatocytes, adipocytes, etc. (2) The presence of sodium orthovanadate during solubilization and receptor purification steps appears to be essential for adequate recovery of the earliest stages of phosphorylation of the insulin receptor or tyrosine residues. (3) It is essential that the wheat germ agglutinin-agarose columns are thoroughly washed with the buffer after samples are applied to assure optimal receptor purification. (4) Incubation of cells with anti-receptor antibodies can be shortened to 2 hr at 4° without any apparent decrease in the amount of receptor that can be immunoprecipitated. (5) If the main interest is in the phosphotyrosine content of the receptors, immunoprecipitation can be carried out with anti-phosphotyrosine antibodies. 33 Antibodies to O-phosphotyrosyl residues are prepared in New Zealand white rabbits by using keyhole limpet hemocyanin conjugate of N-bromoacetyl-o-phosphotyrosine as the antigen. Purification of the antiserum is achieved by applying serum onto a O-phosphotyrosineSepharose column and eluting with 0.2 M nitrophenyl phosphate in 50 mM HEPES, pH 7.4. The eluate is then dialyzed against 50 mM NaCI before use. (Preparations of antibody, antigen, and column are described in Ref. 33.) (6) Insulin receptor phosphorylated in this way can be used for further analysis, including tryptic peptide mapping, phosphoamino acid analysis, or high-performance liquid chromatography. Phosphorylation of the Insulin Receptor in a Cell-Free System Insulin-receptor phosphorylation in cell-free systems is analogous in many respects to the events taking place with intact cells. 32 Material at various stages of purification, from crude plasma membranes to receptors purified almost to homogeneity, have been used for phosphorylation. It appears that the purified insulin receptor is a tyrosine kinase capable of phosphorylating not only the/3 subunit of the receptor but also tyrosine residues of several authentic and synthetic proteins or peptides. The insu33 D. T. Pang, B. R. Sharma, and J. A. Shafer, Arch. Biochem. Biophys. 242, 176 (1985).

[53]

INSULIN AND GROWTH HORMONE RECEPTORS

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lin receptor thus acts as both a protein kinase and a substrate. Insulin activates the kinase activity by increasing the Vmaxof the reaction. Among nucleotides, only ATP is capable of supporting the kinase activity. These properties allow one to separate the roles of the insulin receptor as substrate and as an autophosphorylating enzyme.

Insulin-Stimulated Receptor Autophosphorylation. Preparation of the partially purified insulin receptors: Cultured cells (IM-9 lymphocytes, EBV-transformed lymphocytes, or other mononuclear cells) are harvested (600 g, 10 min, 4°). Cells are resuspended in 0.25 M sucrose, 50 mM HEPES, pH 7.6, 1 mM phenylmethylsulfonyl fluoride, and 1 /zg/ml aprotinin. The cell suspension is homogenized with a motor-driven glass homogenizer at 4° and centrifuged at 600 g for 5 min at 4°. The supernatant is saved and the pellet is rehomogenized and sedimented again at 600 g for 5 min at 4°. The supernatants are then combined. Crude membranes from combined supernatants are obtained by spinning at 20,000 g for 90 min at 4°. The pellet is solubilized in 1% Triton X-100, 50 mM HEPES, pH 7.6, and 1 mM phenylmethylsulfonyl fluoride (4-8 ml) for 60 rain at 4° with constant rotation or shaking. The supernatant is centrifuged at 120,000 g for 45 min at 4°. The resulting supernatant is applied to a wheat germ agglutinin-agarose column (1 ml). The wheat germ agglutinin column is washed with 75 ml of 50 mM HEPES, pH 7.6, 150 mM NaCl, and 0.1% Triton X-100. Glycoproteinenriched material is eluted with two aliquots (1 ml each) of 0.3 M Nacetyl-D-glucosamine in 50 mM HEPES, 150 mM NaC1, and 0.1% Triton X-100. Protein content and specific 125I-labeled insulin binding to each fraction of the column eluates (l-ml fractions) are determined. Phosphorylation assay: The solubilized, lectin-purified cell extracts (enriched for insulin receptor) are incubated without or with insulin (1100 nM final concentration) with 50 mM HEPES, pH 7.6, in a final volume of 60/~l for 30 min at 22°. The phosphorylation reaction is initiated by addition of 15 /zl of a solution containing 250/zM [y-32p]ATP ( - 3 Ci/ /zmol), 25 /zM ATP, 25 mM manganese acetate, and 5 mM CTP. The reaction is terminated in 5-10 min by addition of either 25/~l "stopping" solution, if immunoprecipitation is to be carded out, or 50/zl of "stopping" solution and "sample" (5x concentration)buffer (1:1 mix), if analysis by SDS-PAGE is to follow. The sample is immunoprecipitated with anti-insulin receptor antibodies (final dilution of sera from patients with these antibodies is 1 : 100-1 : 250) for 16 hr at 4°. Immune complexes are precipitated with protein A (Pansorbin). Pellets are washed, resuspended in buffer containing SDS (5%, v/v) and 2-mercaptoethanol (1.82 M), and boiled (100 °, 5 min). Phosphoproteins are analyzed by 7.5% SDS/polyacrylamide gel electrophoresis and autoradiography.

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Comments: (1) Various cell types, including freshly isolated human peripheral blood cells (monocytes, erythrocytes) or cultured lymphocytes or tibroblasts, can be used in these studies to investigate clinically relevant problems. 34(2) Clearly, the more cells that are obtained and the more efficiently they are disrupted, the more material will be available for preparation of the insulin receptor. Sonication of the cell suspension or addition of excess glycerol to the cells (leading to osmotic lysis) can be considered as alternatives to homogenization. (3) Presence of sodium vanadate (to inhibit ATPase activity), dithiothreitol, and glycerol (to stabilize the kinase activity) might be advantageous in optimizing the tyrosine kinase activity of the receptor preparations. (4) Manganese acetate alone can support the phosphorylation reaction. Addition of MgC12 does not seem to increase the insulin effect. (5) For insulin receptor autophosphorylation studies using cells with few receptors, all volumes in this reaction should be scaled up to accommodate the 5- to 10-fold increase in the amount of the receptor preparation necessary for optimal results. (6) Autophosphorylation experiments can also be performed at 4° after a 1- to 16-hr preincubation with insulin. Insulin-Stimulated Phosphorylation of Exogenous Substrates. Phosphorylation assay: Solubilized, lectin-purified insulin receptor preparations (see above) are preincubated for 30 min at 22° in the absence or presence of insulin (final concentration 1-100 nM) in a total volume of 60 Ixl. The exogenous substrate (e.g., casein, histone, amino acid polymers) to be phosphorylated by the insulin receptor kinase is added (usually 40/zl of 10 mg/ml solution). Phosphorylation is initiated by addition of 40/.d of a solution containing 250/~M [y-32p]ATP, 100/zM ATP, and 70 mM MgC12. The reaction is terminated (usually at 5-30 min) by spotting aliquots onto 3 × 3 cm squares of filter paper (Whatman No. 3) and placing them into a bath containing 10% trichloroacetic acid (v/v) with l0 mM sodium pyrophosphate. Filter papers are thoroughly washed (four to six changes of the bath solution) with constant stirring (with a magnetic stirring bar) over 24 hr at room temperature. The filter papers are rinsed twice with ethanol, once with ether (optional), allowed to air dry, and the 32p content determined in a liquid scintillation counter. Comments: (1) The random copolymer consisting of glutamate and tyrosine residues at ratio of 4 : 1 (Sigma) has proved the best substrate for the tyrosine kinase activity of the insulin receptor in all systems studied thus far. (2) Insulin-like growth factor (IGF-I) receptors present in the receptor preparation will also be stimulated by insulin (in excess of l0 -s M) to phosphorylate the Glu : Tyr copolymer. (3) Substrates, insulin, and 34 G. Grunberger, Y. Zich, and P. Gorden, Science 223, 932 (1984).

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TRANSFERRINRECEPTORS

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stock solution of MgCI2 are prepared in 50 mM HEPES, pH 7.6. Sodium vanadate (1-2 mM) can be used in the phosphorylation reaction to inhibit phosphotyrosine phosphatases. Addition of manganese does not appear essential for the tyrosine kinase activity of the insulin receptor in this assay; magnesium alone supports this activity (there is a linear relationship between both basal and insulin-stimulated kinase activities and MgCI2 concentration up to - 4 0 mM). (4) An efficient way to run these experiments is to have sufficient volume of the incubation mixture (e.g., 140/~1) in each test tube for removing two (60/zl) aliquots; the reaction is initiated by adding the [T-32p]ATP, •ATP, and MgC12 mixture to successive test tubes every 30 see; at the end of the additions a 60-/zl aliquot is removed from each successive tube every 30 sec and spotted on filter papers; this maneuver is then repeated for a second 60-/zl aliquot. This maintains constant reaction time for each experimental tube. Thus each tube is sampled at two different time points (e.g., 10 and 20 min). If each condition is set up in duplicate or triplicate, enough data are generated in each experiment to assure reproducibility and to assess the kinetics of the phosphorylation reaction.

[54] L y m p h o i d R e c e p t o r s for T r a n s f e r r i n B y ROLAND A. NEWMAN

Background The transferrin receptor is a transmembrane protein found on the surface of all proliferating cells. The human transferrin receptor has been the most completely characterized 1-3 although the mouse transfen'in receptor appears structurally very similar. 4,5 All cells require iron for growth and transferrin represents the way in which vertebrates have solved the problem of transporting a toxic and an essentially insoluble ion through the circulation. Transferrin receptors present on the cell surface i C. Schneider, R. Sutherland, R. A. Newman, and M. F. Greaves,J. Biol. Chem. 257, 8516 (1982). 2 I. S. T r o w b r i d g e a n d M . B . O m a r y , Proc. Natl. Acad. Sci. U.S.A. 7 8 , 3 0 3 9 (1981).

3R. A. Newman, C. Schneider, R. Sutherland, L. Vodinelich, and M. F. Greaves, Trends Biochem. Sci. 7, 397 (1982). 4 p. A. Stearne, G. A. Pietersz, and J. W. Goding, J. Immunol. 134, 3474(1985). 5A. Van Agthoven, C. Goridis, P. Naquet, A. Pierres, and M. Pierres, Eur. J. Biochem. 140, 433 (1984). METHODS IN ENZYMOLOGY, VOL. 150

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.