Proc. Natl. Acad. Sci. USAVol. 84, pp. 3690-3694, June 1987Biochemistry

Identification of the receptor for erythropoietin by cross.linking toFriend virus-infected erythro-id cells

(erythropoiesis/plasma membranes)

STEPHEN T. SAWYER, SANFORD B. KRANTZ, AND JUDITH LUNADepartment of Medicine, Division of Hematology, Vanderbilt University School of Medicine, and Veterans Administration Medical Center,Nashville, TN 37232

Communicated by Stanley Cohen, February 27, 1987

ABSTRACT Erythropoietin (Epo) is a glycoprotein hor-mone that regulates erythroid development and interacts withsurface receptors' n developing erythroid cells. In this labo-ratory, a cell system with a relatively pure population oferythroid cells that respond to Epo has been developed.Immature erythroid cells are obtained from the spleens of miceinfected with the anemia strain of Friend virus. The binding of1'"I-labeled Epo ('2-I-Epo) to plasma membranes from thesecells was studied in this investigation. '291-Epo binding reachedequilibrium within 20 min at 37TC. Twenty percent of thereceptors bound 125I-Epo with a Kd of 0.08 x 10-9 M, while theremaining receptors bound the hormone with a Kd of 0.6 x10-9 M. In this study, a receptor for Epo was identified bycross-linking 1251-Epo to the receptor in intact cells and plasmamembrane preparations using disuccinimidyl suberate. Poly-acrylamide gel electrophoresis revealed two labeled bands of100 and 85 kDa. The 85-kDa band was more heavily labeled(65%) than the 100-kDa band. Both bands were equallydecreased when increasing-amounts of unlabeled Epo wereincluded in the binding mixture, indicating a specific interac-tion of 12.I-Epo with the receptor.

Erythropoietin (Epo) is a glycoprotein hormone that is theprimary regulator of erythroid maturation (1). We havepreviously studied the binding of radioactive Epo to Friendvirus anemia strain-infected (FVA) erythroid cells (2, 3),which are relatively pure immature erythroid cells thatrespond to the hormone by undergoing late erythroid matu-ration (4-7). During erythroid differentiation, these FVA-infected erythroid cells respond to Epo with globin genetranscription (7), synthesis of hemoglobin (4, 5, 8), transferrmnreceptors (8), erythrocyte-specific proteins including spec-trins, band 3, and band 4.1 (5, 6), and other normal eventssuch as enucleation and extensive membrane rearrangement.Previously, we have shown the binding of 251I-labeled Epo(1251I-Epo), with full biological activity, to 300 higher-affinityreceptors (Kd, 0.09 x 10-9 M) and 650 lower-affinity recep-tors for Epo (Kd, 0.57 x 10-9 M) on the surface of these cells.At 370C, bound 125I-Epo was internalized by receptor-mediated endocytosis and eventually degraded with thesecretion of [125I]iodotyrosine into the medium (2).

Identification and characterization of various receptorshave been probed with cross-linking agents that covalentlyattached radioactive ligands to the receptor. One of the morecommonly used bifunctional cross-linkers is disuccinimidylsuberate (D$S), which has been used to detect such receptorsas those for insulin (9), nerve growth factor (10), type ptransforming growth factor (11), platelet-derived growthfactor (12), angiotensin II (13), and others. In the presentstudy, the receptor for Epo was identified by cross-linking1251I-Epo bound to receptors on intact FVA-infected cells and

plasma membranes from these cells with DSS. In addition,the binding of I251-Epo to the plasma membrane fraction wascharacterized.

MATERIALS AND METHODSMaterials. Human recombinant Epo was obtained from

AMGen Biologicals (Thousand Oaks, CA). Na125I was ob-taimed from Amersham. DSS and Iodo-Gen (1,3,4,6-tetra-chloro-3a,6a-diphenylglycouril) were obtained from Pierce.Friend virus, pseudotype SFFVA/FRE cl-3/MuLV(201)originally obtained from W. D. Hankins at the NationalInstitutes of Health, was maintained by the passage ofinfectious plasma in BALB/c mice.

Cells and Plasma Membrane Preparation. Immature ery-throid cells were purified from the spleens of CD2F1 miceinfected with FVA by velocity sedimentation at unit gravitythrough a continuous gradient of bovine serum albumin asdescribed (4, 14). To prepare plasma membranes from FVA-infected erythroid cells, the total spleen was disrupted to asingle cell suspension and the erythrocytes were lysed byexposure to 150 mM NH4Cl/15 mM Tris HCl, pH 7.65, at37TC for 30 s. The NH4CI/Tris!HCl solution was diluted 1:4with Iscove's modified Dulbecco's medium and the cellswere pelleted by centrifugation at 500 x g for 15 min. Thepellet was resuspended in NH4Cl/Tris HCl and the proce-dure was repeated. The cells were then washed in 150 mMNaCl and 10mM Tricine (pH 7.4) three times and resuspend-ed in 10 mM KCI/10 mM Tris HCl, pH 7.4, containing 1 mMEGTA and leupeptin (1 Ag/ml). After homogenization by 10strokes with a Dounce homogenizer, the homogenate wasfractionated by differential centrifugation and the plasmamembrane fraction was recovered after equilibrium sedimen-tation on a discontinuous sucrose gradient as described byothers for erythroleukemia cells (15). About 10 mg of plasmamembrane protein was obtained from this procedure startingwith five mice.

Iodination of Epo. Epo was iodinated using Iodo-Gen (16)(2 Axg), which was coated on the walls of a conical reactionvial. Epo (1.3 Ag of protein) and 200 gCi of 125I (1 Ci = 37GBq) were incubated in the reaction vial for 5 min at roomtemperature in a final vol of 50 al of 0.5 M phosphate buffer(pH 7.0) containing 0.02% Tween 20. After the incubation,the contents of the reaction vial were transferred to a tubecontaining 10 mg of KI in phosphate-buffered saline, 0.1%bovine serum albumin, and 0.02% Tween 20; 1251-Epo wasseparated from the free 1251 by chromatography over aBio-Gel P6 column. This procedure provided Epo with0.3-1.0 molecule of 1251 per molecule (20-75 ttCi per ,ug ofprotein) and with full biological activity. Biological activitywas assayed by the ability of 125I-Epo to stimulate hemoglo-bin synthesis in FVA-infected erythroid cells as determined

Abbreviations: Epo, erythropoietin; 125I-Epo, 125I-labeled Epo;FVA, anemia strain of Friend virus; DSS, disuccinimidyl suberate.

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by incorporation of 59Fe into heme (14). Increasing concen-trations of 1251-Epo and Epo gave parallel and superimpos-able curves of increasing 59Fe heme concentration.

Binding and Cross-Linking of 12-5-Epo. 1251-Epo was incu-bated with 10-40 Ag of protein of the plasma membranefraction at 370C for the time indicated in 100 mM phosphatebuffer (pH 7.4) containing 1 mM EGTA, unless otherwiseindicated, and 0.1% bovine serum albumin. The bindingmixture was then applied to 0.2-,um Millipore filters andwashed with 10 ml of phosphate-buffered saline containing0.1% bovine serum albumin. The filters were then counted ina y counter. Nonspecific binding was determined in thepresence of 100 units of Epo per ml and was subtracted(nonspecific binding was 15-25% of total binding at saturat-ing level of 1251-Epo).

125I-Epo was incubated with intact FVA-infected erythroidcells (5 units/ml) at 370C for 30 min in Iscove's modifiedDulbecco's medium containing 1% bovine serum albumin.The incubation mixture was transferred to an ice bath andDSS was added to a final concentration of 0.2 mM. After 15min of incubation in the cold, the cross-linking reaction wasquenched by the addition of TrisHCl (pH 7.4) to a finalconcentration of 50 mM, and the cells were pelleted bycentrifugation at 1000 x g for 5 min. The cells were resus-pended in 50 mM Tris HCl (pH 8.0) containing 100 mM NaCland washed two times. The cell pellet was resuspended in0.1% Triton X-100 and 20 mM Hepes (pH 7.4) and disruptedby mixing in a Vortex. The nuclei were pelleted by centrif-ugation at 500 x g for 3 min and the supernatant wascombined with sample buffer and boiled for 3 min. Thismaterial was analyzed by NaDodSO4/PAGE as described byLaemmli (17), and dried gels were exposed to Kodak XAR-5film.

125I-Epo was bound to receptors on the plasma membranefraction and was cross-linked in a similar fashion as intactcells. Membrane protein (1 mg) was incubated with 7.5 unitsof 125I-Epo per ml in 100 mM phosphate buffer (pH 7.4)containing 1 mM EGTA and 0.1% bovine serum albumin for15 min at 37°C. The mixture was transferred to an ice bath,and DSS was added to a final concentration of 200 ,uM for 15min unless otherwise indicated. Tris HCl (pH 8.0) was addedto a final concentration of 150 mM, and the membranes werepelleted by centrifugation at 13,000 x g for 30 min. The pelletwas resuspended in 50 mM Tris HCl, pH 7.4/100 mM NaCland washed twice. The pellet was suspended in sample bufferand boiled for 3 min. The material was analyzed by NaDod-S04/PAGE as described above.

RESULTSThe binding of 1251-Epo to the plasma membrane fraction ofFVA-infected erythroid cells required 10-20 min at 37°C toreach equilibrium (Fig. 1). When binding was carried out inthe absence of proteinase inhibitors, the binding of 1251-Epoincreased to a maximum at 20 min and then declined.However, the inclusion of 1 mM EGTA in the bindingmixture prevented the decline of 125I-Epo bound to theplasma membrane, suggesting that calcium-activated pro-teinase activity degraded the receptor for Epo or the hormoneitself.The binding of 1251-Epo to a particulate fraction of a whole

cell lysate of FVA-infected erythroid cells revealed 5.8 x 106receptors per ,g of protein (data not shown). Binding of125I-Epo to the plasma membrane fraction of these cells gavea value of 42.4 x 106 receptors per ,ug of protein (Fig. 2).While this represents a 7.3-fold increase in the receptors forEpo per /ig of membrane protein, the -fold purification wasless than expected if all the receptors were predominantly inthe plasma membrane. Examination of the binding of 25I-Epo to other membrane fractions showed that both the

TIME, min

FIG. 1. Binding of 125I-Epo ('25I-EP) to the plasma membranefraction of FVA-infected erythroid cells. Ten units of 251I-Epo per mlwas incubated with 20 ,ug of membrane protein for the indicated timeat 37°C in the absence (o) or presence (e) of 1 mM EGTA in thebinding buffer. Specific binding was determined as described. Dataare the means of duplicate determinations.

lysosomal membrane and rough endoplasmic reticulum frac-tions had -20 x 106 receptors per ,ug of protein. This is notunexpected, as our previous work has shown that '25I-Epo isinternalized and degraded within the cell (2). Therefore, thetransit of the receptor within the cell compartments should bereflected in the wide distribution of the receptors for Epoamong all the membrane fractions.

Binding was carried out at different concentrations of125I-Epo as shown in Fig. 2. Saturation of the binding sites forEpo on the plasma membrane occurred at a concentrationbetween 7 and 10 units of Epo per ml. When these data wereanalyzed by the method of Scatchard (Fig. 2 Inset), twoclasses ofbinding sites for Epo were evident. Twenty percentof the receptors have a higher affinity for Epo (Kd, 0.08 x10-9 M) than the remaining receptors (Kd, 0.6 x 10-9 M). Thebinding constant was calculated on the reported values of 30kDa as the molecular mass and 128,000 units per mg of Epo

ox.c0)2a-

Eau

zD0m01'

N

125 -EP BOUND pM

2 5 7 10125I-EP, Units/ml

15

FIG. 2. Effect of concentration of 251I-Epo (1251I-EP) on thebinding to plasma membranes from FVA-infected erythroid cells.Membranes (20 ,ug of protein) were incubated for 30 min at 37°C withthe indicated concentration of 251I-Epo. Specific binding was deter-mined as described. (Inset) Data replotted by the method ofScatchard. B/F, bound/free.

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glycoprotein (18). The binding activity in these membraneswas stable when stored at -80TC for >6 months.The cross-linking of 125I-Epo to FVA-infected cells and

plasma membrane fractions from FVA-infected cells resultedin the generation of two major radioactive bands that migrat-ed in gels containing reducing agent to positions correspond-ing to a molecular mass of 135 kDa and 120 kDa as shown inFig. 3. Other minor bands are also present at lower molecularmass. None of the bands was found when cross-linking wascarried out in the presence of 100 units per ml of unlabeledEpo as shown in lanes B, D, and F. The molecular mass ofthe bands corresponded to 100 kDa and 85 kDa when the35-kDa contribution of Epo was subtracted. Epo has amolecular mass of 30 kDa but migrates at an apparentmolecular mass of 35 kDa on NaDodSO4/PAGE due toglycosylation (18). In lane C, the 85-kDa band was labeledwith 231 cpm and the 100-kDa bands contained 140 cpm of1251. Together, this radioactivity represents 0.7% of the totalspecifically bound I251-Epo.To observe the cross-linked bands in the intact cell exper-

iment (Fig. 3, lane A), the gel was exposed to film 10 timeslonger than the gel from the plasma membrane experiment(lanes B and E). Since the results were identical using theintact cells and the plasma membrane fraction, all additionalexperiments were carried out using the plasma membranefraction. When the cross-linked plasma membrane samplewas analyzed on NaDodSO4/PAGE containing no reducingagent, both the 100-kDa and 85-kDa bands were seen (laneE).The effect of the concentration of DSS on the cross-linking

of 125I-Epo to the membrane was examined (Fig. 4). Increas-ing the concentration ofDSS from 10 ,uM to 500AM increasedthe radioactivity found in both the 100-kDa and 85-kDacross-linked proteins up to 1% of specifically bound 125I-Epo.A greater level of labeling of the 85-kDa protein compared tothe 100-kDa protein (65:35) was observed at every concen-tration of DSS tested. At 1 mM DSS or greater, a largefraction of the labeled material no longer entered the gel anddiscrete protein bands on the gel became very diffuse. Thus,we selected 0.2 mM DSS as an optimal concentration ofcross-linking reagents.The effect of time on the cross-linking of 125I-Epo to the

plasma membrane fraction of FVA-infected cells using 0.2mM DSS was examined. As shown in Fig. 5, exposure ofDSSfor 5 min at 4°C was sufficient for 125I-Epo to be cross-linkedto the 100-kDa and 85-kDa proteins. Additional incubation

[ DSS]pyM500 200100 50 20 10

200

118

92

_f "m

*#+? ::A.C D

A 8 C D E F

FIG. 4. Effect of the concentration ofDSS on the cross-linking of125I-Epo to plasma membranes from FVA-infected erythroid cells.Cross-linking was carried out as described except the concentrationofDSS was varied. The material was analyzed by NaDodSO4/PAGEon 5% acrylamide gels and by autoradiography. Size markers are inkDa.

did not substantially increase the radioactivity cross-linked tothe major proteins or to minor lower molecular weightproteins, nor did it affect the ratio of 125I-Epo cross-linked tothe 100-kDa and 85-kDa proteins. In the absence of DSS, noradioactivity of a molecular mass greater than 125I-Epo wasfound (lanes G and H).

Increasing concentrations of unlabeled Epo were added tothe binding cross-linking reaction to examine the nonspecificcross-linking of 125I-Epo to the plasma membrane fractionfrom FVA-infected erythroid cells. As shown in Fig. 6, theradioactivity in the 100-kDa and 85-kDa bands was reducedby the addition of unlabeled Epo in equal proportion. More-over, the multiple bands of cross-linked material presentbetween 85 kDa and 35 kDa were also reduced in proportionby the presence of increased levels of unlabeled Epo. Thisresult shows that the nonspecifically bound 1251-Epo, which

Exposure to DSS, min

5 15 30 0

200200

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-118 - 1

92

D::" aE:

c D E F

FIG. 3. Cross-linking of 'l25-Epo to plasma membranes and intactFVA-infected erythroid cells. 125I-Epo was bound to intact cells(lanes A and B) and plasma membranes (lanes C-F) and cross-linkedwith 0.2 mM DSS as described. Binding was carried out in thepresence of 200 units of unlabeled Epo per ml (lanes B, D, and F) andits absence (lanes A, C, and E). The cross-linked material wasanalyzed by NaDodSO4/PAGE on 5% acrylamide gels in thepresence of2-mercaptoethanol (lanes A-D) and absence (lanes E andF). The positions of molecular size markers (in kDa) are shown.

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35

A B C D E F G H

FIG. 5. Effect of time on the cross-linking of 125I-Epo to plasmamembranes of FVA-infected erythroid cells. 125I-Epo was bound toplasma membranes as described, in the presence (lanes A, C, E, andG) or absence (lanes B, D, F, and H) of 100 units of unlabeled Epoper ml. DSS (0.2 mM) was added for 5 min (lanes A and B), 15 min(lanes C and D), or 30 min (lanes E and F). No DSS was present inreplicates incubated for 30 min (lanes G and H). The samples werethen subjected to NaDodSO4/PAGE on 7.5% acrylamide gels and byautoradiography. Size markers are in kDa.

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FIG. 6. Effect of unlabeled Epo on the cross-linking of '"I-Epoto plasma membranes from FVA-infected erythroid cells. Plasmamembranes were incubated with 5 units of 125I-Epo and no unlabeledEpo (lane A), or 0.5 (lane B), 1 (lane C), 2 (lane D), 5 (lane E), 10 (lane

F), 20 (lane G), 50 (lane H), or 100 (lane I) units of unlabeled Epo per

ml. Cross-linking was carried out as described. The material was

analyzed by NaDodSO4/PAGE on 7.5% acrylamide gels and byautoradiography. Size markers are in kDa.

is 15-20% of the total bound 125I-Epo, does not appreciablycontribute to the 1251-Epo cross-linked to membrane proteins,and the minor bands of '251-Epo-labeled proteins result fromthe cross-linking of specifically bound Epo.

DISCUSSIONBinding of 125I-Epo to plasma membranes from FVA-infectedcells is very similar to the binding of the hormone to intactcells, both in the time course of binding and in the concen-tration of Epo required to saturate the binding sites. Thehigher-affinity receptors survive the disruption of the cellsand purification of the membranes by equilibrium sedimen-tation. Approximately 25% of the total receptors have a

higher affinity than the remaining receptors on both intactcells (2) and plasma membranes (Fig. 2). These membranesmaintain stable binding sites for 125I-Epo for >6 months whenstored at -80'C, so this material can be accumulated over aperiod of time to provide a large source of receptors for Epo.The receptor for Epo may be sensitive to calcium-depen-

dent endogenous proteinase activity as the binding of 1251_Epo declines during incubation at 370C. This effect seems tobe due to the degradation of the receptor rather than 1251-Eposince preincubation in the absence ofEGTA and then bindingin the presence of EGTA results in a reduction in 125I-Epobinding (data not shown). Other workers have shown that theresponsiveness of cells to Epo is greatly reduced by thedigestion of the cell surface with proteinase (19). The inclu-sion of EGTA to chelate calcium bound to the membraneseliminates the degradation of the binding sites and demon-strates that divalent cations are not required for the bindingof 1251-Epo to its receptor. This is in contrast to previouswork, which implied that calcium was required for thebinding of Epo to mouse erythroid progenitor cells (20).

Cross-linking of 125I-Epo to both intact FVA-infectederythroid cells and plasma membranes from these cellsresults in the labeling of two major proteins of 100 kDa and85 kDa. Because of the low number of receptors for Epopresent on these immature erythroid cells (800-1500) and theinefficiency of cross-linking the heavily glycosylated Epo,the detection of cross-linked proteins was greatly facilitatedby the use of plasma membranes over intact cells. Thelabeling of two separate proteins by 125I-Epo raises thequestion ofhow these proteins are arranged in the membranein relation to the binding site(s) for Epo on the receptor. We

cannot distinguish if the binding site requires both proteins.It is also possible that '25I-Epo is cross-linked to a nonrecep-tor protein that is in close proximity to the binding site forEpo on the receptor. Cross-linking to 1251I-Epo bound tomembranes at a range of increasing concentrations resultedin the labeling of both the 100-kDa and 85-kDa bands in thesame ratio of 35:65 and increased labeling of both bands inparallel with the binding of both high- and low-affinity sites(data not shown). Cross-linking of 125I-Epo to erythroleu-kemia cells that have only low-affinity receptors (2) gives thesame ratio of 35% 100-kDa protein to 65% 85-kDa proteinlabeled with 125I-Epo (data not shown). These findingssuggest that the high- and low-affinity receptors are notrelated to the existence of the 100-kDa and 85-kDa proteins.However, the possibility that one of the proteins labeled with25I-Epo is not a receptor protein is not easily tested.It is possible that the 85-kDa protein cross-linked to

'25I-Epo is derived by cleavage of the 100-kDa polypeptide orthat both proteins are derived from alternative posttransla-tional modification of a common precursor. However, thereis some evidence that proteinase activity in the plasmamembrane fraction is not responsible for the generation ofthe85-kDa protein from the 100-kDa cross-linked protein duringthe binding and cross-linking reaction. This was initiallyconsidered a strong possibility because of the observationthat the binding of 1251-Epo to plasma membranes reached aplateau and then declined drastically at 370C (Fig. 1). Varyingthe time of 125I-Epo binding at 370C by using membranesprepared in the presence or absence of proteinase inhibitorsand comparing intact cells versus plasma membranes (Fig. 3)in the cross-linking reaction invariably resulted in the sameratio of labeling of the 100-kDa protein compared to the85-kDa protein. This suggests that the 85-kDa protein is notderived from the cleavage of the 100-kDa protein. However,experiments are now under way to examine whether the85-kDa protein is derived from the 100-kDa protein bypeptide mapping. The broad range of less-abundant proteinscross-linked to 125I-Epo in the molecular mass range from 35to 115 kDa was greatly increased in membranes not contain-ing inhibitors of proteinases, and they are likely degradationproducts of the 100-kDa and 85-kDa proteins.

In conclusion, the present study shows that plasma mem-brane Epo receptors can be prepared from the FVA-infectederythroid cells and that these Epo receptors are identical inbinding properties to those in intact cells. Cross-linking of125I-Epo to the membranes and intact cells results in thelabeling of 100-kDa and 85-kDa proteins that are eithersubunits of the receptor for Epo, multiple forms of thereceptor, the receptor and a modified receptor, or thereceptor plus a neighboring protein. Additional experimentsare required to confirm which of the above possibilities istrue.The identification of the receptor for Epo by the cross-

linking of 125I-Epo has been preceded by the characterizationof most of the receptors for hematopoietic factors. Twogeneral categories of these receptors have now becomeapparent: the receptor for granulocyte colony-stimulatingfactors (G-CSF) and macrophage colony-stimulating factor(CSF-1 or cfms protooncogene) are large (150-160 kDa)single-chain peptides, which have some structural similarityto the receptors for epidermal growth factor (EGF) andplatelet-derived growth factor (PDGF) (12, 21, 22). However,receptors for interleukins 2 and 3 (IL-2, IL-3, or multi-CSF)and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to be either single species or multiplenon-disulfide-bonded receptors of much smaller molecularmass-51-75 kDa (reviewed in ref. 21). Although the molec-ular mass of the receptor for Epo is intermediate betweenthese two categories, the multiple cross-linked bands of the

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receptor for Epo are similar to the structure of the receptorsfor IL-2 and IL-3. IL-3 has been cross-linked to 55-kDa and75-kDa proteins (23), and IL-2 has been cross-linked to a55-kDa receptor and an apparent regulatory protein of 70kDa, which seems to be necessary for high-affinity binding(24). The receptors for Epo and IL-3 are similar in that thetwo IL-3 cross-linked proteins (55 kDa and 75 kDa) appar-ently are independent of high-affinity versus low-affinitybinding of IL-3. The mechanism(s) through which thesesmaller receptors for factors such as IL-2, IL-3, GM-CSF,and Epo transmit the surface signal to the interior of the cellare unknown. It will be ofgreat interest to follow the progressof these investigations to see whether structural and func-tional similarities such as the similarities in the receptors forEGF, PDGF, insulin, and CSF-1, which are tyrosine-specificprotein kinases, are found in these small molecular massreceptors for hematopoietic factors.

We wish to thank AMGen Biologicals for information on the natureof the recombinant Epo and access to unpublished data. This workwas supported by National Institutes ofHealth Grants AM-15555 andT32 AM-07186 and by the Veteran's Administration.

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