Binding Sites for Lactogenic and Somatogenic Hormones from

THE JOURNAL OF BIOLOGICAL CHLMISTRY Vol. 258, No. 1, Issue of January 10, pp. 305-314, 1983 Printed in U. S A .

Binding Sites for Lactogenic and Somatogenic Hormones from Rabbit Mammary Gland and Liver THEIR PURIFICATION BY AFFINITY CHROMATOGRAPHY AND THEIR IDENTIFICATION BY IMMUNOPRECIPITATION AND PHOTOAFFINITY LABELING’

(Received for publication, May 17, 1982)

Marie-Theres HaeuptleS, Michel L. Aubertg, Jean Djianel, and Jean-Pierre KraehenbuhlS From the $Institute of Biochemistry, University of Lausanne School of Medicine, 1066 Epulinges, §Department of Pediatrics and Genetics, University of Geneva School of Medicine, 1211 Geneva, Switzerland, and flLaboratoire de Physiologie de la Lactation. Institut National de la Recherche Agronornique, Centre National de la Recherche Scientifique, 78350 Jouy en Josas, France

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Lactogenic and somatogenic hormone-binding sites from rabbit liver or mammary membranes, solubilized with the nonionic detergent Triton X-100, were purified by affinity chromatography. The procedure takes advantage of the high affinity between biotin and streptavidin, a nonglycosylated biotin-binding protein. Bio- tin was covalently bound to human growth hormone, a ligand which binds both to lactogenic and somatogenic hormone-binding sites. Biotinylated human growth hormone was first allowed to interact in solution with the solubilized binding sites. The complexes formed were then adsorbed to insolubilized streptavidin and were subsequently eluted with 5.0 M MgClz or sodium dodecyl sulfate. The radiolabeled binding site that was purified from mammary membranes migrated as a 35- kilodalton band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas two liver binding sites were resolved as a major 67-kilodalton and a minor 35-kilodalton band. An antireceptor antibody, which inhibits the binding of lactogenic hormones, precipitated the 35-kilodalton band from mammary membranes and to a lesser extent the same band from liver, while the liver 67-kilodalton band was not recognized by this antibody. Thus, the liver and mammary 35-kilodalton bands were identified as lactogenic binding sites. Photoaffinity labeling of solubilized mammary binding sites, using human growth hormone derivatized with ethyl-4-azidophenyl-1,2-dithiobutyr- imidate confirmed the apparent M, = 35,000 found by affinity chromatography. Liver membrane proteins were solubilized with sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred onto nitrocellulose and incubated with radioiodinated human growth hormone. The radiolabeled hormone labeled the liver 67-kilodalton band, indicating that this polypeptide corresponds to the growth hormone- binding site. In addition to the monomeric binding sites for Iactogenic and somatogenic hormones, multimers of the binders were observed under nonreducing conditions. Similarly, aggregates were revealed when solubilized liver or mammary membranes incubated with

li This work was supported by Grant 3.672.0-80 from the Swiss National Science Foundation, a grant from Nestle Nutrition, and Grant 166.A.K.80.1 from the Swiss League against Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer-

this fact. tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate

radioiodinated human growth hormone were chromatographed on Sephadex G-200. This suggests that both the lactogenic and growth hormone-binding sites form muktimer complexes in the membrane which can be cross-linked via disulfide bridges.

Specific binding sites for lactogenic hormones, z.e. prolactin, placental lactogen, primate growth hormones, are expressed in various tissues of vertebrates (1). The hormones have been implicated in the regulation of numerous activities in a number of biological systems, although in most cases, a direct correlation between binding of the hormone to a receptor and expression of the biological response has not been established. In addition, it is not known whether there is a common primary mechanism whereby lactogenic hormones exert their effects.

The biological system that has been most studied with regard to the mechanism of action of lactogenic hormones is the control of lactogenesis in the mammary gland. Lactogenic hormones appear to be the primary regulators of milk protein (caseins and a-lactalbumin) gene expression (2-10). They induce an accumulation of casein mRNA by increasing its synthesis and extending its half-life (7) . The binding of the hormones to a cell surface receptor on mammary epithelial cells is required for the biological response to occur (ll), and the interaction of the hormones with the plasma membrane receptor has been well characterized (12-14). In addition, the receptor has been shown to respond both to down- and up- regulation by lactogenic hormones (15, 16). Little is known, however, about the cellular or molecular events which allow the lactogenic hormones to exert their effect on milk protein gene expression, and attempts to ident,ify possible mediators have thus far failed (17).

There is increasing evidence that polypeptide hormones, including insulin (18-21), epidermal growth factor (22, 23), nerve growth factor (24, 25), and prolactin (14, 26, 27) enter the cell by endocytosis and are eventually found associated with nuclear structures. Based on such observations, it has been postulated that internalization is a necessary step to allow dispatching of hormones to cellular sites involved in the biological response (20, 22, 26, 27). It was also proposed that internalization of the hormone-receptor complex was followed by lysosomal degradation of the ligand, the receptor, or both to form secondary messengers (22, 28-30). Using Iysosomo- tropic agents, which interfere with lysosomal function, and microfilament-disrupting drugs, Houdebine and Djiane (31) suggested that endocytosis and the subsequent lysosomal

305

306 Binding Sites for Lactogenic and Somatogenic Hormones

degradation of the hormone-receptor complex were not essen- tial for the activation of casein gene expression. More recently, an antibody directed against a partially purified lactogenic hormone receptor was shown to induce casein mRNA accumulation when added to cells at low concentrations, whereas at high concentrations, it inhibited casein synthesis (32). These results are consistent with the hypothesis that the binding of lactogenic hormones to its receptor induces confor- mational changes of the receptor or surrounding membrane components, which in turn trigger the formation of mediators. Furthermore, the agonist effect of these antibodies rules out a direct interaction of the hormone with chromatin structures. In addition, Teyssot et al. (33) identified a second messenger which is released when plasma membranes containing lactogenic hormone receptors are incubated with lactogenic hormones or antibodies against the receptor and which initiates transcription of specific milk protein genes in isolated nuclei from mammary tissue. It is possible that such a mediator is a small peptide cleaved from the receptor itself. Thus, it becomes important to purify the receptor by affinity chromatography using specific ligands, z.e. lactogenic hormones or antibodies, and to compare its structure with that of the native receptor isolated with probes lacking agonist activity.

In this study, we describe a new affinity chromatography procedure in which a biotinylated hormone that has interacted in solution with its corresponding binding site is adsorbed to the insolubilized biotin-binding protein, streptavidin. Using hGH' as the ligand, which has been shown to bind both to the lactogenic and the somatogenic receptors (34), we have been able to purify to homogeneity both binders and to compare their binding properties to those of the binding sites from native or solubilized membranes. By photoaffinity labeling, immunoprecipitation using anti-prolactin receptor antibodies, and electrophoretic transfer of liver membrane proteins onto nitrocellulose followed by blotting using radioiodinated hormones, we confirm that the lactogenic and somatogenic binding sites are distinct molecular entities.

EXPERIMENTAL PROCEDURES~

Material R e s *ere Obtdlned frm the follo*lng source$

LdCtOperwida Ie (punf led g'ade 100 Uirnq) and 3-jGP-chola.~d~propyl) dvmthyl- ~ O n O J - I - p ~ O p d n e - I ~ I f ~ ~ ~ t ~ 2HzO (CHAPS) frm Calh~ochrm, Sari Dleqo. Calif.; chloramne-l and d i t h i o t h r e 3 t a l f r a Herck, Oamrtadt , Germany; r a b b l t g a m - g l o b v i l n s ( f rac t ran 1 1 ) from Miles Laboratories lnc . Xankdkee. 111.; bo17ne i e r m dlbmnn. dloxane and l r l t o n X-100 from FIUka, Bvchr , Sr l t ie r land: ge la t ln and p n e n y l ~ t h y l l u l f ~ n y l - f i u o r l d e (QnSF) frm Serva, Heidelberg, Gemany; peps- t l t i n A . t l -a i ty lg lucorlde (I-0-n-o~tyl-a-D-qlucopyranorlde), and Iodlum salt O f deoxycnollc acld from Slgma, St-LOUIS. no i A f f l g e l - I 0 frm Blo-Rad Labold-

Burllngame. Cll7f.i Sephadex and Prote ln A-Sephdrole frm P h d m C l a Flne Cheml- t o r l e l , Richmond. C a l l f . ; agarose-bound av ld ln 0 from Vector Laboratories I n c . ,

c a l l . Uppsdld, Sweden, polyethylene qlycol 6000 f ra Slegf r led , lo f lngen , Sul- t i e r land , n l t roce l lu lore sheets . 0 45 ym pore s12e. ~n r o l l f o m from W l l l ~ p o r e Corp . Bedford, M d l s . Colodlm bags for Y ~ C Y Y ~ d l a l y z l l frm Sartorius, Goeftln gen, Gemany. NaiZ51 ;nd O-carboo~ l - I (C -b~Otro f i m Pmerrhm. Bu<k>nghamrh~re,

Compmy, Pockford. I l l . . StTeptawd3n was a generous g l f t f r a n 0.5 . Papermaster England. e thy l r -a2 ldapheny l - l . 3 -d l th ,~b" tyr lnr lda te . HCl from Plerce Chmlca l

( v a l e UrllYeTslty)

Hamanes Ovine prolactin (OPRL) INIH-PS I1 and 1 2 ) was Ohtalned through the hormone d l s t n b u t ? a n proqram of tne N d t l o n d l l n s t l t u t e o f Arthrltll and Wetabo- 11c D ~ s e d s e s , Bethesdd, Md. P u r l f l e d 0v1ne p m l a c t l n and bowne growth h o m n e

U n l v e r l l t y o f C a l ~ f a r n l a . San F P ~ C I I C O ) . Human growth hormone (hGH) ( N L ~ o T - (bGH) were generously ~ u p p l l e d by Or C . H 11 (Hornone Research Laboratory,

.oneR) vas obtalned frm Nardlrl. l n r u l l n I abordtor les . Gentoffe (Denmark)

I The abbreviations used are: hGH, human growth hormone; bGH, bovine growth hormone; CHAPS, 3-[(3-~holamidopro~~l)dimeth- y~ammonio]-l-propane sulfonate; EADB, ethyl 4-azidophenyl-1,3-di- thiobutyrimidate.HC1; oPRL, ovine prolactin; PMSF, phenylmeth- ylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

Portions of this paper (including parts of "Experimental Proce- dures" and ''Results'' and Figs. 2 and 4) are presented in miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-1285, cite authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

Animals-Outbred 4- to 5-month-old New Zealand rabbits were used in all experiments. The rabbits were subcutaneously injected with 2 mg of bromocryptine at midpregnancy and 36, 24, and 12 h prior to removal of the mammary glands. The rabbits were killed 2 days before parturition or during early lactation.

Methods

Radioiodination-oPRL, hGH, and bGH were radioiodinated either using lactoperoxidase and 1251 (carrier-free) (35) or by a modi- fication of the chloramine-T method (36), including in the final reaction mixture (50 p l ) 1 mCi of NaiZ51, 5 pg of hormone in 0.1 M Na phosphate buffer, pH 7.3, and 4 pg of chloramine-T. The reaction was stopped after 1 min by addition of 10 p1 of 1% Na metabisulfite and 100 p1 of a solution containing 1% KI, 10% sucrose, 0.1% sodium azide, and 250 pg of bovine serum albumin. Aggregated hormone and free iodine were separated from radioiodinated monomeric hormone by gel filtration on Sephadex (3-100.

Preparation of Mammary and Liver Membrane Fractions-Dis- section, homogenization, and fractionation of mammary gland and liver tissues from late pregnant or early lactating animals were performed as described elsewhere (37). Protease inhibitors, including PMSF at 1 mM final concentration and pepstatin A at 0.2 pg/ml final concentration, were added to the homogenates.

Detergent Solubilization of Membrane Proteins-The membrane suspensions were dialyzed against 50 mM Tris-HC1, pH 7.6, containing 0.1 M NaC1,lO mM MgC12, 0.02% sodium azide or were ultracentrifuged (2 h a t 200,000 X gnus), and the pellets were directly suspended in the same buffer. In order to determine the optimal conditions for solubilization, various amounts of Triton X-100, sodium deoxycholate, or a mixture of both was added to the membrane suspension. Alterna- tively, 30 mM P-octylglucoside, 10 mM CHAPS, or 2% SDS was used. The samples were agitated for 30 min a t room temperature. Insoluble material was removed by centrifugation for 2 h a t 200,000 X g,,,. Generally, membrane suspensions containing a protein concentration of 1 mg/ml for mammary gland and 5 mg/ml for liver yielded, upon solubilization with 0.5 Triton X-100, about 50% of the original protein concentration in the high speed supernatant.

Protein Determinations-These were performed according to the method of Lowry et al. (38) with 0.1% SDS in the reaction mixture. Bovine serum albumin dissolved in the same reaction mixture served as standard.

Siluer-staining Procedure-Some SDS-polyacrylamide gels were silver stained following the procedure of Wray et al. (39). The sensi- tivity of the procedure was assessed using bovine serum albumin as a standard and as little as 0.5 ng of protein was still detected on the gel.

Binding Assays-Hormone-binding activity was detected using methods previously described (12-14). Since oPRL forms aggregates in the presence of Triton X-100 (13), all binding studies were carried out with hGH. Briefly, 100-200 pg of particulate membrane proteins, or 20-100 pg of detergent-solubilized membrane poteins were incubated with 0.1-0.2 ng of 1'511-labeled hormone (2 X IO' cpm) in a total volume of 0.5 ml of 25 mM Tris-HC1 buffer, pH 7.6, containing 10 mM MgC12, 0.5% bovine serum albumin, 0.02% NaN.] in the presence or absence of unlabeled hormone (0.1-200 ng) for 16-24 h a t room temperature. The reaction was stopped by adding 2 ml of ice-cold buffer (4 "C) to particulate membranes, or 0.5 ml of buffer containing 0.1% (w/v) rabbit y-globulin instead of bovine serum albumin followed by 1 ml of cold 25% (w/v) polyethylene glycol 6000 to solubilized membranes. In order to separate free hormone from membrane or polyethylene glycol-precipitated hormone-binder complexes, the tubes were centrifuged a t 3000 X gaUa at 4 "C for 30 min, the super- natants were discarded, and the tubes were drained and counted in a Packard y-counter. In each assay, nonspecific binding was determined in control tubes with an excess (2 pg) of unlabeled hormone added to the incubation mixture.

Kinetic and Statistical Analysis-Analysis of the binding data was performed on a CDC Cyber 170 computer using the Scatfit analysis program developed by Munson and Rodbard (40).

Gel Filtration-Detergent-solubilized membranes (1.5 ml) were chromatographed on a Sephadex G-200 column (1 X 100 cm) equilibrated with 50 mM Tris-HC1, pH 7.6,O.l M NaCI, 10 mM MgC12,0.02% NaNs (column buffer), containing 0.1% Triton X-100. The column was calibrated with blue dextran, rabbit IgG, human transferrin, ovalbumin, chymotrypsinogen A, hGH, cytochrome C, and N a T as molecular weight markers. The eluted fractions were assayed for lactogenic hormone-binding activity, and the data were analyzed by Scatchard plot, Alternatively, 0.5 ml of Triton X-100-solubilized mem-

Binding Sites for Lactogenic and Somatogenic Hormones 307

branes was fist incubated with 5 X IO6 cpm of "'I-hGH in the presence or absence of excess (100 pg) unlabeled hormone followed by gel filtration on the same Sephadex G-200 column. Aliquots (100 p l ) of the eluted fractions were counted in a y-counter. Finally, some detergent-solubilized membranes (1.5 ml) were incubated with 50 pmol of dithiothreitol/mg of protein for 2 h at room temperature and were chromatographed on Sephadex G-200, and eluted fractions were assayed for binding activity. Those tubes that bound "51-hGH were pooled, concentrated to 200 pl by vacuum dialysis, incubated with 5 X lo6 cpm of I2'I-hGH, and applied to the same column. The eluted fractions were then counted.

Affinity Chromatography Biotin Labeling of Hormones-Biotinyl-N-hydroxysuccinimide es-

ter was synthesized using ['4C]biotin and was cross-linked to hormones as described (41). The binding properties of biotinylated hormones were analyzed using a radioreceptor assay (13, 14) and were compared to native hormone. If the number of biotin residues per hGH hormone molecule was lower than 20, the binding properties were not significantly altered.

Znsolubilized Streptauidin-Affi-Gel 10 (2 mlj, washed once with isopropanol and three times with cold KnO, was incubated 4 h at room temperature with 0.6 mg of streptavidin in 1 ml of 0.1 M NaHCO.3 and IO6 cpm of '"1-streptavidin to monitor the coupling efficiency. The beads were then incubated 2 h at room temperature with 2.0 M glycine buffer, pH 9.0, to quench free reactive groups. About 0.2 mg of streptavidin was coupled per ml of packed Affi-Gel.

Isolation of Hormone-binding Sites-One ml of Triton X-100- solubilized membranes was incubated overnight a t room temperature with 25 pg of biotinyl-hGH and 10' cprn of "'I-biotinyl-hGH in a final volume of 1.15 ml. The hormone-binder complex was precipitated using 350 pl of 0.1% rabbit y-globulin as carrier and 1.5 ml of 25% (w/ v) polyethylene glycol 6000 and was centrifuged for 30 min at 3000 x g.,,a at 4 "C. The resulting pellet, resuspended in column buffer containing 1% Triton X-100, was allowed to stand for 16 h a t 4 "C in order t.o solubilize the hormone-binder complex, and then was incubated 15 min at 4 "C with 50 p1 of streptavidin-Affi-Gel (or agarose- bound avidin D) with constant stirring. The beads were pelleted by centrifugation and were washed three times with 1% Triton X-100 in column buffer, three times with 0.1% Triton X-100 in column buffer, and three times with 30 mM P-octylglucoside in column buffer. Elution of the binding sites was achieved either with 2 times 100 $1 of 20 mM Tris-HCI buffer, pH 7.6, containing 5.0 MgCla, 30 mM P-octylglucoside, or 1.0 M glycine-NaOH buffer, pH 10, containing 2% SDS. When MgCI2 eluates were assayed for hGH-binding activity, they were diluted with 25 mM Tris-HCI, pH 7.6, containing 10 mM MgCL, 0.5% bovine serum albumin, 0.02% NaN:1 and were dialyzed overnight against the same buffer in order to lower the MgCL concentration.

Iodination of the Isolated Binding Sites-Binding sites eluted with 5.0 M MgC12 were iodinated using the chloramine-T technique with 50 pl of eluate, 0.5 mCi of Na? in 5 pl, and 35 p1 of chloramine- T (2 mg/ml), and a 30-s incubation time. The reaction was stopped by adding 20 p1 of 1% Na metabisulfite and 35 pl of 1% KI.

Binding sites eluted with SDS were iodinated using the iodine monochloride procedure for 30 s: 150 p1 of eluate, 0.5 mCi of Na'""1 in 5 pl, and 50 p1 of IC1 stock solution diluted 1 : l O with 2 M NaCl (42). In both procedures, free "'I was separated from labeled protein on a Sephadex G-25 column equilibrated with 20 mM Tris-HCI buffer, pH 7.5, containing either 0.2% SDS or 0.005% rabbit y-globulin, 0.1% Triton X-100.

Immunoprecipitation of Purified Hormone-binding Sites-Bind- ing sites were purified from 4 ml of solubilized mammary or liver membranes using biotin-streptavidin affinity chromatography. The SDS-eluted material was radioiodinated by the iodine monochloride procedure (42). Samples containing 2-3 X loH cpm were run on 10% SDS-PAGE, and the bands corresponding to the M, of the lactogenic or growth hormone-binding sites were excised and eluted by electro- dialysis (ISCO sample concentrator Model 1750) in 300-400 pl of 0.01 M Tris-HC1 buffer, pH 8.3, containing 0.01% SDS. The eluted material contained approximately 1% (2 X lo6 cpm) of the originally applied material. Samples (50,000 cpm) were incubated with increasing concentrations of a goat serum directed against partially purified lactogenic hormone receptor from rabbit mammary membranes (32) or with preimmune serum as control. In parallel experiments, '"I-hGH was incubated with the same immune reagents. After 16 h of incubation at 4 "C, a sheep serum directed against goat IgG was added at a dilution of 1:5 (-0.2 mg/ml of specific antibodies), and the mixture was further incubated for 24 h at 4 "C. The immune complexes were

recovered by centrifugation at 3,000 X g,,, for 30 min, and the resulting pellets were counted in a y-counter. Alternatively, 0.8 X 10' cpm of purified radiolabeled binding sites were incubated overnight a t 4 "C with antireceptor serum diluted 1:125 in a final volume of 0.5 ml. For controls, some samples were preincubated with a 10-fold excess of cold binding sites eluted from streptavidin-Affi-Gel 10.

The immune complexes were adsorbed onto 5 pl of packed protein A-Sepharose, were washed and eluted as described elsewhere (43j, and were then analyzed by SDS-PACE and autoradiography.

Photoaffinity Labeling Deriuatization of hGH-The coupling of the cleavable heterobi-

functional cross-linking reagent EADB to hGH was modified from Lewis et al. (44). hGH (0.6 mg) in 0.5 ml of 0.1 M NaHC0:r was mixed with 100 p1 of 27.3 mM EADB in the same buffer and was stirred at room temperature for 60 min. To separate unbound EADB from the hormone, the reaction mixtures were dialyzed against 10 mM sodium phosphate buffer, pH 7.0 (2 liters), overnight a t 4 "C. Derivatized hormones were stored at -20 "C. Iodination using the chloramine-T procedure was performed as described.

Covalent Binding of Photoreactive "'I-hGH to Solubilized Hor- mone- binding Sites-One hundred pl of supernatant from Triton X- 1 0 0 (0.5%)-solubilized membranes (50 pg of protein) were incubated with 10' cpm of EADB-"'I-hGH for 12-15 h at room temperature in the dark. Control samples were preincubated overnight at room temperature with an excess of 20 pg of unlabeled hGH. The reaction mixtures were photolyzed at 4 "C for 15 min using a Hanovia 450- watt high pressure mercury lamp equipped with a Corning No. 3220 glass filter. The hormone-binder complexes were precipitated with polyethylene glycol, and the pellets were resuspended in 50 mM N- ethylmorpholino-HCI buffer, pH 7.3, containing 2% SDS and were allowed to stand for 2 h at room temperature. Residual Triton X-100 and lipids were extracted by ethanol precipitation of the proteins, and the resulting pellets were analyzed by SDS-PAGE and autoradiography.

Blotting of Hormone- binding Sites-Liver membranes solubilized with 2% SDS in the presence of proteinase inhibitors were centrifuged 5 min in an Eppendorf table centrifuge and then were electrophoresed on SDS-polyacrylamide gels. Electrophoretic transfer of the proteins from the SDS-polyacrylamide gels onto nitrocellulose paper was performed according to Towbin et al. (45). The electrophoretic blots were soaked with 0.5% gelatin and 0.1% Triton X-100 in column buffer (gelatin solution) for at least 2 h at room temperature to allow renaturing of the hormone-binding sites and saturation of additional protein-binding sites on the nitrocellulose paper. The blots were then incubated with 12.5 X 10' cpm of "'I-hGH in 25 ml of gelatin solution by gentle agitation for 15 h at room temperature. Control samples were preincubated with 5 pg of cold hGH/ml of gelatin solution. Finally, the blots were washed twice with gelatin solution, twice with 0.1 M Tris-HCI buffer, pH 7.5, containing L.5 M NaC1, 10 mM MgCI,, 0.028 NaN.$, twice with 10 mM Tris-HC1 buffer, pH 7.6, and once with distilled water, followed by drying onto filter paper. The blots were finally exposed for autoradiography.

SDS-PAGE-Polyacrylamide gel electrophoresis in the presence of SDS was performed using a 10% polyacrylamide concentration unless otherwise stated (46).

RESULTS

Solubilization of Hormone-binding Sites-In order to de- fine the optimal solubilization conditions, membrane fractions from late pregnant or early lactating mammary glands of rabbits were treated with increasing concentrations of Triton X-100 (0.05-3.0%), sodium deoxycholate (0.05-1.5%), or a combination of both detergents. The hGH-binding capacity and the affinity constants as estimated by Scatchard plot analysis as well as the amount of protein recovered were determined both in a high speed supernatant and the pellet. The best results were obtained when mammary membranes at a protein concentration of 0.5-1 mg/ml were treated with 0.25-0.5% Triton X-100 (Fig. 1). Under such conditions, 70% of the membrane proteins were solubilized and 65% of the hGH- binding sites were recovered in the supernatant. Increasing the detergent or the membrane protein concentration im- proved neither the amount of membrane protein solubilized


nor the number of binding sites detected in the supernatant. The amount of detergent-insoluble proteins recovered in the pellet decreased with increasing concentrations of detergent. A significant amount of binding sites (20-50%) remained associated with the detergent-insoluble material even at high detergent concentrations. The sum of the number of binding sites recovered in the supernatant and the pellet never ex- ceeded the number of sites determined in equivalent amounts of nonsolubilized mammary membranes. Therefore, detergent treatment did not expose masked binding sites, which is in agreement with published reports (47). A 5-fold increase of the association constant followed membrane solubilization with 0.58 Triton X-100 (Table I), a finding which has been reported by several investigators (12,13). The affinity constant decreased by 25% with higher Triton X-100 concentrations. Sodium deoxycholate alone or in combination with Triton X- 100 allowed solubilization of more membrane proteins (85%) but reduced by half the number of binding sites detected in the supernatant (Fig. 1). The amount of binding sites solubilized with various detergents including Triton X-100, CHAPS, and P-octylglucoside as well as the affinity constants are given in Table I. Since Triton X-100 or other detergents containing

phenol groups interfere with the radioiodination procedure, P-octylglucoside was tested and shown to preserve the binding capacity of the solubilized binding site, although only a third of the binding sites solubilized with Triton X-100 were recovered with 30 mM ,8-octylglucoside. When membranes were solubilized in 2% SDS, followed by gel filtration in the presence of 0.1% Triton X-100, about 60% of the binding activity was recovered, and the affinity constant of hGH for the renatured binding site remained similar to that estimated from Triton X-100-treated membrane.

Gel Filtration of Solubilized Hormone-binding Sites-Tri- ton X-100-solubilized liver or mammary membranes were incubated to equilibrium with '"I-hGH, and the mixture was chromatographed on a Sephadex G-200 column under reducing or nonreducing conditions (Fig. 2). Under all conditions, the radioactive material eluted in two main peaks, the first one containing the hormone-binder complexes and the second one the unbound hormone. The radioactivity was displaced from the first peak by excess unlabeled hormone, with a concomitant increase in the free hormone radioactivity. '"I- hGH-binder complexes from mammary membranes eluted as a large peak corresponding to an average A4,. = 200,000 (Kay

% Trlton X-lo0 a DOC ' Y s i T r i t m X-100

protein conc.(mglml)

FIG. 1. Detergent solubilization of lactogenic hormone-binding sites from mammary membranes. A constant amount of mammary membranes (0.5 mg of protein/ml) was treated with 0.05-38 Triton X-100 ( A ), 0.05- 1.5% sodium deoxycholate (DOC) ( B ) , or a combination of 1% Triton X-100 and 0.05-1.58 deoxycholate (C). Alternatively, 1% Triton X-100 was added to a mammary membrane suspension, the protein concentration of which varied from 0.5-5 mg/ml (D). The recovery of the amount of protein (open bars) and the hGH-binding capacity (shaded bars) was based on the values obtained with mammary membranes before solubilization (100%). hGH- binding capacity and affinity constants ( K A ) were estimated by Scatchard plot analysis. The values given for K A represent the mean of the values obtained for the different detergent concentrations +- S.E.

TABLE I Binding capacity and affinity constants of hGH and bGH for mammary or liver binding sites

The affinity constant KA (X IO9 "I) and the binding capacity Q were estimated by Scatchard plot analysis. Q is expressed as nanograms of bound hGH and represents the recovery of binding sites based on 1 mg of total protein in the particulate membrane suspension. Except for liver particulate membranes, where a high affinity low capacity, l) , and a low affinity high capacity, 2), binding site was detected, low affinity high capacity sites were not found in any other preparation. Mean values of three independent experiments I+_ S.E. are given for particulate or Triton X- 100-solubilized membrane preparations, and for MgCI, eluates from Triton X-100 or CHAPS-solubilized membranes. ND, not determined.

Liver

hGH bGH Sample hGH in mammary gland __

KA 8 K4 8 K4 Q Particulate membranes (microsomes) 6.8 f 0.8 2.6 +. 0.6 I) 2.95 k 0.5 17.8 -r- 5 I ) 7.4 f 2.3 6 i 0.8

2) 0.09 f 0.07 560 f 519 2) 0.3 f 0.2 950 f 900 Triton X-100-solubilized membranes 36 f 4 2.1 f 0.1 4.35 f 1.0 18.6 & 11 8.1 f 4.1 3 f 1.4 CHAPS-solubilized membranes 17.4 2.6 3.6 f 0.6 32.6 & 3.5 6.1 f 1.2 4.5 * 0.65 p-Octylglucoside-solubilized membranes 42 0.8 ND ND ND ND MgCl, eluate from Triton X-100-solubilized mem- 26 f 5 0.26 f 0.14 4.6 f 0.6 2.8 & 1.2 0.65 f 0.40 3.4 f 1.6

MgCL eluate from CHAPS-solubilized membranes 25 0.29 3.4 8.1 1.8 2.1 branes

= 0.15), with a shoulder corresponding to the void volume (Fig. 2 A ) . In the presence of dithiothreitol, the material which eluted with the void volume was shifted to the first peak which became sharper (Fig. 2B). About 20% of the total radioactivity was recovered with the first peak when 30 ng of '"I-hGH were incubated with 0.5 ml of solubilized membrane proteins. Detergent-solubilized membranes were also chromatographed in the absence of Iz5I-hGH, and the eluted fractions were assayed for hGH-binding activity by Scatchard plot analysis. As shown in Fig. 2 A , the hGH-binding activity coincided with the first radioactive peak. Similar elution pro- files were obtained with solubilized liver membranes, although the first peak eluted with an average M, - 300,000 (Fig. 2C).

Purification of Hormone-binding Sites-An affinity chromatography procedure was developed in which the ligand derivatized with an active ester of biotin was first incubated with solubilized hormone-binding sites, and the hormone-

binder complexes were subsequently adsorbed to insolubilized streptavidin. Since biotinylated hGH was used as the ligand, it was expected that both the lactogenic and somatogenic hormone-binding sites from liver membranes would be adsorbed, but only the lactogenic hormone-binding site from mammary membranes, assumptions which were based on the results reported by Shiu and Friesen (13) and Waters and Friesen (34). The binding sites eluted from the mammary membranes (Fig. 3A) migrated under both reducing and nonreducing conditions as a 35-kilodalton band. In the presence of dithiothreitoi, the high M , aggregates disappeared. The 23- kilodalton band co-migrated with biotinylated hGH. To assess for specificity, either the membranes were incubated with nonbiotinylated hormone, or the insolubilized streptavidin was presaturated with free biotin. Except for monomeric (17 kilodaltons) and a small amount of tetrameric (68 kilodaltons) streptavidin, no bands were visualized. Identical results were obtained when the binder was purified from microsomes solubilized with CHAPS (data not shown). When hGH binding sites from liver membranes were isolated and analyzed by the same procedure, a major M, = 67,000 band appeared (Fig. 3B), the apparent M, of which did not change upon reduction and alkylation. A faint band, barely visible in Fig. 3B but apparent on overexposed gels, co-migrated with the 35-kilodalton binding site isolated from mammary membranes. In controls analogous to those performed with mammary membranes, only monomeric and polymeric streptavidin were identified.

Binding Properties of the Purified Binding Sites-Table I summarized the binding activities of the MgC12 eluates from affinity columns and compares these activities to those of particulate or solubilized membranes. A single class of binding sites was estimated for hGH in purified mammary binder preparations, with an affinity constant (KA = 10'" M") similar to that found for solubilized membranes, but approximately s-foid higher than that of part,iculate membranes. This change in affinity was observed with all detergents tested. In contrast, no significant change in the association constant of hGH for purified liver binder preparation or solubilized liver membranes was observed when compared to the constant estimated for particulate membranes, while the constant for bGH binding to the purified liver binding sites decreased -5- to 10- fold. TWO classes of binding sites were estimated in particulate membranes irrespective of the ligand used, whereas detergent treatment of liver membranes abolished binding of both hGH and bGH to the low affinity high capacity binding site. In liver membranes or in purified liver binding site preparations, radioiodinated hGH was displaced more efficiently by cold hGH than by cold bGH (Fig. 4). The amount of hGH hound/ mg of liver membrane protein as estimated by Scatchard plot analysis was -10-fold higher than that bound by mammary membranes. The recovery of purified binding sites from mammary membranes calculated from Table I was in the range of 10% of the original binding activity and for liver binding sites in the range of 30%. The concentration of lactogenic hormone binder in the microsomal fraction from late pregnant rabbits treated with bromocryptine was -100 fmol/mg of protein (-4 ng mg" protein), a value which is in good agreement with published data (12). When the mammary binding site purified from 1 mg of microsomal membrane protein was resolved by SDS-PAGE (Fig. 3 ) and the polyacrylamide gel was stained with the silver-staining procedure (39), no band was stained in the position of the 35-kilodalton binding site, although the procedure allowed the detection of 0.5 ng of serum albumin (data not shown). Based on the binding studies of Table I, one can calculate that 1 mg of microsomal membrane proteins contains -4 ng of lactogenic hormone-binding site. Since the


A. MAMMARY GLAND 0. LIVER

z 136 - 1"

80- 68-

80- 68-

43 - 43-

a b c a b c a b c a b c

- D l l + DTT - DTT + DTT

FIG. 3. 10% SDS-PAGE of hGH-binding sites affinity purified from mammary or liver plasma membrane-enriched preparations. hGH-binding sites were purified from Triton X-100-solubilized mammary ( A ) or liver ( B ) membranes by incubation with biotinylated hGH and subsequent extraction of the hormone-binder complexes using streptavidin-Affi-Gel 10 as described under "Methods" (a) . Controls consisted of incubating the samples with unbiotinylated hGH ( b ) or presaturating streptavidin-Aff-Gel 10 with excess free biotin (c). The material eluted with 2 8 SDS from the insolubilized streptavidin was radioiodinated by the iodine monochloride (42) procedure and analyzed by SDS-PAGE under reducing (+dithiothreitol (DTT)) or nonreducing (-dithiothreitol) conditions. When reduced, the samples were alkylated with iodoacetamide. The band which migrates close to the front of the gel represents monomeric streptavidin. Much less monomeric form is eluted from the adsorbent presaturated with free biotin (lanes c), probably because biotin stabilizes tetrameric streptavidin. The scale represents M, X lo-''.

. I . . ... 9.. ... ,.I . .... ,;. ;. ,.I ,.I

recovery of our purification procedure is lo%, only 0.4 ng of binder can be expected to migrate in the position where the 35-kilodalton band is detected by autoradiography as shown in Fig. 3A. Such a concentration is below the detection sensi- tivity of the silver-staining procedure. Therefore, it appears that the degree of purification of the mammary binder ap- proaches the theoretical value of -28,000.

Immunoprecipitation of the Purified Binding Sites-Puri- fied binding sites from mammary or liver membranes were immunoprecipitated as described under "Methods" using a goat serum directed against partially purified prolactin receptors (32). The antiserum contained antibodies which were shown to prevent the binding of prolactin to receptors present in membranes of various tissues. In addition, the antibodies exhibited lactogenic activity in that they were able to release from membranes that contained prolactin receptor a second messenger which in turn triggered the transcription of specific milk protein genes in isolated mammary nuclei (48). The

binding sites purified from liver or mammary membranes by biotin-streptavidin affinity chromatography and electrodi- alyzed from SDS-polyacrylamide gels were precipitated with serial dilutions of the goat anti-prolactin receptor serum. The titration curves using the 35-kilodalton binding site as antigen indicated a low titer of specific antibodies in the antiserum. Both the mammary 35-kilodalton band and to a lesser extent the liver lower band were specifically immunoprecipitated, while the liver 67-kilodalton band was not recognized by the anti-prolactin receptor serum (Fig. 5). These experiments indicate that lactogenic hormone-binding sites are present both in liver and mammary membranes and share antigenic determinants which are not expressed on the liver 67-kilodalton band.

Hormone- blotting Experiments-In order to identify further the liver 67-kilodalton binding site, electrophoretic blotting was used to correlate hGH-binding activity to protein bands resolved by SDS-PAGE. Receptor-binding activity was shown to be restored after SDS treatment (SDS-PAGE) by exchanging this detergent with Triton X-100. Radioiodinated hGH labeled intensively the M, = 67,000 liver band, as well as higher M , bands and material which did not enter the gel (Fig. 6). Since reduction and alkylation abolished the hormone- binding activity of the liver binding site, it was not possible to determine whether the higher M , material represented multimers of the M , = 67,000 band. However, preincubation of the blots with excess cold hGH efficiently abolished the labeling of the M, = 67,000 band but also of the higher M, bands, suggesting that the latter represented aggregated hGH- binding sites. Since the 67-kilodalton band was not immunoprecipitated by the anti-prolactin receptor serum, we conclude from the blotting experiments that the liver 67-kilodalton binding site is likely the somatogenic hormone-binding site.

Photoaffinity Labeling of Solubilized Lactogenic Hor- mone-binding Sites-To test whether the affinity chromatography procedure we have developed efficiently extracted all hGH-binding entities from solubilized mammary membranes, we photoaffinity-labeled solubilized mammary binding sites, and analyzed the photocross-linked products by SDS-PAGE.


100.

8 0 -

60 .

40.

2 0

136.

80. 68.

43

L

A B C D

FIG. 5. Immunoprecipitation of hormone-binding sites purified from mammary or liver membranes. Radioiodinated 35- kilodalton hGH-binding sites from mammary and liver membranes as well as the 67-kilodalton binder from liver were eluted from SIX- polyacrylamide gels. Eluted hGH-binding sites (8 X 1 0 cpm) were incubated with a goat serum directed against partially purified prolactin receptors from rabbit mammary membranes (32) at a final dilution of 1:125 in the absence or presence of excess unlabeled purified binding sites. Immune complexes were adsorbed to protein A-Sepharose and analyzed by SIX-PAGE (top) or counted in a y- counter (bottom). Top, lanes a-c, radiolabeled purified 35-kilodalton mammary binding site. Lanes d-i, radiolabeled purified 35-kilodalton (d-f) and 67-kilodalton ( g - i ) liver binding sites. Lanes a, d, and g. purified binding sites prior to immunoprecipitation. Some degradation products are present. Lanes b. e, and h, immunoprecipitations with anti-prolactin receptor serum. Lanes c, and i, immunoprecipitations in the presence of excess unlabeled binding sites eluted from streptavidin-Affi-Gel 10. The scale represents M , X 10 :I. Bottom, the radiolabeled 35-kilodalton mammary (A ) and liver ( B ) binding sites and the 67-kilodalton liver receptor (C) were incubated with antireceptor serum in the absence (open bars) or presence (shaded bars) of excess unlabeled binding sites eluted from streptavidin-Affi-Gel 10, adsorbed to protein A-Sepharose, and counted. In D, equal amounts of liver 35-kilodalton and 67-kilodalton material were incubated as described. Maximal radioactivity was immunoprecipitated from the mammary 35-kilodalton binding sites, yielding between 1 and 2% of the original input (8 X 10" cpm) in three independent experiments. The results shown represent the mean values of these independent experiments, expressed as per cent of maximal immunoprecipitation obtained with the mammary binding sites.

The labeling patterns were then compared with those obtained from affinity chromatography eluates. The cross-linking capacity of the aryl-azide derivative of hGH (EADB-"'I- hGH) was assessed by photoaffinity labeling anti-hGH anti-

26

14

EADB

00- t

6s-

43 -

26-

a b a b a b -DTT +DTT

FIG. 6 (left). Hormone blotting of liver plasma membranes. SDS-solubilized liver plasma membranes (150 pg of protein/slot) were electrophoresed on a 10% SDS-polyacrylamide gel and electrophoret- ically transferred to nitrocellulose. After quenching of additional protein absorption sites on nitrocellulose and renaturation of the liver hormone-binding sites in a 0.5% gelatin, 0.1% Triton X-100-containing buffer, the blots were incubated with ""I-hGH in the absence ( a ) and presence ( b ) of a 1000-fold excess of unlabeled hGH. In the control, the labeled band which migrated slightly faster than the 67-kilodalton band corresponds to bands intensively stained by Coomassie blue which are considered as nonspecifically labeled material. The scale represents M , X 10 ,'I.

FIG. 7 (right). 10% SDS-PAGE of photoaffinity-labeled solubilized mammary membranes. Triton X-100-solubilized mammary membranes (100 pl) were incubated in the dark to equilibrium with 10" cpm of EADB-"'I-hGH (EADB) , in the absence ( a ) or presence ( b ) of 20 pg of unlabeled hGH. After elimination of unbound hormone by polyethylene glycol precipitation and extraction of Triton X-100 and lipids by ethanol precipitation, half of the photoaffinity-labeled material was reduced and alkylated (+DTT, dithiothreitol) and compared to the unreduced (-DTT) samples by SDS-PAGE, followed by autoradiography. The scale represents M , X IO-".

bodies which were then analyzed by SDS-PAGE. Heavy and light chains, as well as IgG molecules, were labeled and exhibited a slower electrophoretic mobility accounted for by an additional M , = 22,000 band corresponding to hGH. Prein- cubation with excess unlabeled hGH abolished binding and, hence, photolabeling of the specific antibodies (data not shown). EADB-"'I-hGH was photocross-linked to solubilized mammary membranes. The elution profile on Sephadex G- 200 columns of the photolabeled binding sites was similar to that shown on Fig. 2 A . The photoaffinity-labeling pattern on 10% SDS-PAGE is illustrated in Fig. 7. A major band with an apparent M , = 57,000 was assumed to represent the smallest entity of a hormone-binding site complex. If the contribution by the M , of hGH was subtracted, M , = 35,000 was obtained for the binding site which corresponded to the M , of the binding site purified by biotin-streptavidin affinity chromatography. A distinct M , = 80,OOO band was also labeled, as well as higher M , material. Preincubation of the membranes with unlabeled hormone prior to photolysis with EADB-I2'I- hGH abolished the labeling of the main M, = 57,000 and 80,OOO bands as well as that of the higher M , material. Some bands (30 kilodaltons), corresponding to bands intensively stained with Coomassie blue, remained labeled in the presence of excess hormone and were considered as nonspecificdy labeled material. When the cleavable EADB-"'I-hGH was cross-linked to the binding site and then reduced and alkyl-


ated, the labeling of the M , = 57,000 band and the higher M , multimers disappeared as expected.

DISCUSSION

The purification of lactogenic and somatogenic hormone- binding sites from rabbit mammary gland and liver described in this study is based on an affinity chromatography procedure which allows the interaction of a biotin-derivatized ligand with its binding site in a soluble phase, followed by the adsorption of the ligand-binder complex to insolubilized streptavidin. To purify the lactogenic or somatogenic binding sites from liver and mammary membranes, we chose hGH as the ligand, a hormone which in the rabbit possesses both lactogenic and somatogenic hormone-binding activity (34, 49-51). It is generally accepted that rabbit mammary gland tissue contains a single class of hGH-binding sites assumed to represent lactogenic sites (12, 14), whereas there is disagreement as to the exact nature of the hGH-binding sites in the liver of female rabbits. Waters and Friesen (34) found by competition studies and Scatchard plot analysis two types of liver binders, a growth hormone-binding site which bound bGH with high affinity and ovine prolactin with low affinity, and a lactogenic site which bound the latter hormone preferentially. This is further supported by a more recent study (49), in which two types of liver binding sites were separated by lectin and affinity chromatography, one of which bound bGH and hGH and was referred to as the somatogenic receptor, and a second lactogenic site which bound exclusively hGH. On the other hand, Cadman and Wallis (50) in a careful study showed that liver membranes from late pregnant rabbits contained very few specific lactogenic binding sites of the type found in the mammary gland. In their study and in complete agreement with Shiu and Friesen (12, 13), they showed that mammary binding sites bound radiolabeled oPRL and hGH which were displaced by unlabeled lactogenic (oPRL and hGH) but not somatogenic (bGH) hormones. Discrepancies between the results of those different groups could be explained by meth- odological differences, especially in detergent concentrations or variations in the rabbits used, since it has been shown that there is an increase in hepatic binding sites during pregnancy due to an increase in somatogenic rather than lactogenic sites (52, 53).

Using our biotin-streptavidin affinity chromatography procedure and hGH as the ligand, a single protein, with an apparent M , = 35,000 was isolated from mammary membranes, while from liver, two binding species were eluted with apparent M , = 67,000 and 35,000. The identification of the mammary 35-kilodalton binding site was made possible by the use of an antiserum directed against partially purified mammary lactogenic receptors. This antiserum has been shown to abolish the binding of lactogenic hormones to mammary membranes and to elicit a lactogenic response similar to that induced by lactogenic hormones (32, 48). Thus, it contains a population of antibodies which recognizes the lactogenic binding site. Since the two 35-kilodalton proteins from liver and the mammary membranes were specifically immunoprecipitated by the antiserum, we conclude that they represent the lactogenic binding site. The apparent M, found for the mammary lactogenic binder is only half of that reported by Waters and Friesen (34). In order to rule out proteolytic degradation of a higher M , binding site, mammary membranes treated with protease inhibitors before detergent solubilization were photoaffinity labeled with a cleavable heterobifunctional photoreactive reagent (EADB) cross-linked to hGH. A major 57- kilodalton band was specifically labeled. The M , of this band, after subtracting the contribution of the M , = 22,000 EADB- hGH, corresponds to the M , of the purified lactogenic binding

site and represents the smallest binding entity. Several labeled higher M, bands likely represent aggregates of the binder since all these bands disappeared when membranes were preincubated with cold hGH prior to photolysis with EADB- hGH. Recently, a 35-kilodalton protein has been isolated by affinity chromatography from mouse liver membranes using insolubilized ovine prolactin and the zwitterionic detergent CHAPS (54). In rat liver, a protein of similar M , was identified as the lactogenic site by photoaffinity labeling (55). The 67- kilodalton liver protein did not react with the anti-receptor antibodies. This band, however, was strongly stained with radioiodinated hGH in hormone-blotting experiments, The 67-kilodalton protein, therefore, likely represents the somatogenic hormone-binding site.

In contrast to the apparent M, found for the lactogenic and somatogenic hormone-binding sites by SDS-PAGE, much higher M , values were estimated by gel filtration for both the liver (300,000) and mammary (200,000) binding sites. In addition, it was not possible to separate by Sephadex G-200 chromatography the two liver binding sites. The M , of the mammary hormone-binder complex estimated by gel filtration is similar to that reported by Shiu and Friesen (12), but higher than the M, - 100,000 found by Jaffe (56). The 200,000 value obtained by Sepharose (12) or Sephadex gel filtration is based on the assumption that the shape and partial specific volume of the lactogenic hormone binder were identical with those of the protein standard used to calibrate the columns. In his study, Jaffe (56) has characterized the hydrodynamic properties of the lactogenic hormone binder from rat liver membranes. The size and the weight of the binder-detergent complex and the amount of detergent bound were determined by gel fitration and sedimentation velocity in sucrose-water and sucrose-deuterated water gradients. The M, found by this approach after subtracting the contribution of the hormone remains higher than that estimated for the purified binder by SDS-PAGE, suggesting that the binding sites are aggregated in the membranes.

Aggregates of the lactogenic or somatogenic binders were observed when purified binding sites were resolved on SDS- polyacrylamide gels under nonreducing conditions. Similar multimers were also revealed by photoaffinity-labeling or hormone-blotting experiments. Reduction of the preparation with dithiothreitol significantly decreased the amount of aggregates with a concomitant increase of binder monomers, suggesting that the hormone-binding sites are cross-linked via disulfide bonds in the cell membrane. Artifactual aggregation, however, generated by detergent treatment or radioiodination required for the identification of the binding sites cannot be ruled out. In other systems, aggregation or clustering of cell surface receptors has been shown to play a crucial role in the expression of the ligand-induced biological activity (57-62). On liver or mammary cells, the topographical distribution of lactogenic and/or somatogenic receptor has not been investi- gated and it is not known whether receptor clustering eventually mediated by disulfide bond formation is required for a physiological rsponse to occur in response to hormonal stim- ulation.

In this study, we provide biochemical evidence for the existence of a single lactogenic binding site in mammary membranes and of two types of binding sites in liver, namely, a lactogenic site similar to the mammary lactogenic binder and a somatogenic site. The MgCI2 elution procedure required for maintaining the receptor-binding activity did not allow US

to recover separately the two liver binders. Based on the assumption that hGH recognized both lactogenic and somatogenic binding sites, while bGH binds exclusively to the latter, we expected that competition studies and Scatchard


plot analysis on a purified mixture of both types of binders containing well defined protein entities would confm the biochemical evidence for a major somatogenic and a minor lactogenic binding site. Accordingly, two binding sites for human, but only one for bovine growth hormone, should have been observed throughout the purification procedure, and the relative amount of bGH binding should have increased. In contrast, particulate liver membranes exhibited two types of binding sites for each of the two hormones (Table I), while solubilization with various detergents and subsequent purification of the binders abolished both low affinity high capacity binding sites. The relative amount of specific bGH binding increased upon solubilization and purification of the binding sites when hGH was used as the affinity probe. Thus, our binding studies substantiate only partially our biochemical data. Drawbacks might result from the fact that heterologous (hGH, bGH, oPRL) rather than homologous (rabbit PRL and growth hormone) hormones have consistently been used for binding studies as well as for affinity chromatography purification. This is supported by the recent work of Cadman and Wallis (50), showing drastic differences in the capacity of rabbit growth or lactogenic hormone to displace lZ5I-hGH when compared to their human, ovine, or bovine counterparts. In addition, they reported a significant difference in the binding to liver membranes of the two lactogenic hormones hGH and oPRL which in rabbit mammary glands have been shown to exhibit identical kinetic and functional properties (12, 14, 50).

The affinity chromatography procedure we have developed uses streptavidin, a nonglycosylated 68,000-dalton protein with a neutral isoelectric point isolated from Streptomyces auidinii (61, 62). It consists, like the egg white protein avidin, of four identical 17-kilodalton subunits, each containing a single high affinity (KA = IOl4 M") biotin-binding site. Avidin, even when highly succinylated in order to decrease the net positive charge, binds in the absence of biotinylated ligand to liver membranes. It has been suggested that nonspecific binding to liver membranes was due to surface carbohydrate on avidin (63). Similarly, in our study, avidin-agarose nonspecifically adsorbed nonbiotinylated hormone-binder complexes. In addition, the binding of biotinylated hormone-binder complexes could not be prevented by presaturating avidin-agarose with biotin, in contrast to what was found with insolubilized streptavidin. Thus, the absence of a carbobydrate moiety and lower isoelectric point make streptavidin the preferred matrix for specific adsorption of any biotinylated ligand-receptor complex, particularly when present as a minor membrane constituent. When a ligand is linked directly or via a spacer to insoluble matrix, as is the case in conventional affinity chromatography techniques, it becomes difficult to evaluate the biological activity of the insolubilized ligand and whether binding activity has been impaired or completely lost (13). In our procedure, the binding capacity as well as the biological activity of the biotinylated ligand can be determined inde- pendently in a fluid phase and compared to the one of the native molecule. Furthermore, the number of biotin molecules cross-linked to the ligand can be controlled by varying the stoichiometry of the ['4C]biotinyl-N-hydroxysuccinimide ester added to the ligand. In the case of hGH, the coupling of up to 20 biotin molecules to the hormone did not significantly alter the affinity constant of the hormone-receptor interaction when compared to the underivatized hormone. A further advantage of using biotinylated ligand is that the binding sites can be visualized at the cell or tissue level with either strop- tavidin coupled to fluorochromes for light microscopy or to colloidal gold for electron microscopy (64). Therefore, the same biotinylated ligand can serve first to characterize the

binding and biological properties of the derivatized ligand in a fluid phase, second to purify the binding sites by affinity chromatography with insoluble streptavidin, and finally to correlate these biochemical parameters with the subcellular, cellular, or tissue distribution of the receptor.

Acknowledgments-We wish to thank Dr. P. A. Kelly who kindly provided us with anti-prolactin receptor serum and Esther Schaerer who has performed the immunoprecipitation assays. We are grateful to Dr. D. S. Papermaster from the Yale University School of Medicine who has purified streptavidin, Dr. C. H. Li who kindly provided us with highly purified bGH, and Dr. M. Zachmann (University of Zurich) who gave us large amounts of purified hGH (clinical grade).

We thank Dr. Richard Rodewald for helpful criticism and revision of the manuscript and Margot Korn and Z. Freiwald for skilled preparation of the manuscript.

1.

2. 3.

4.

5.

6.

7.

8.

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10.

11.

12.

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19.

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21. 22.

23.

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26. 27.

28. 29. 30. 31.

32.

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