Purification and characterization of recombinant human activin B

Biochimica et Biophysica Acta, 1039 (1990) 135-141

135

Elsevier BBAPRO 33685

Purification and characterization of recombinant human activin B Charles H. Schmelzer 1, Louis E. Burton 1, Cathleen M. Tamony 1, Ralph H. Schwall 2, Anthony J. Mason 3 and Nanette Liegeois 1 t Recooery Process R & D, 2 Pharmacological Sciences and 3 Deoelopmental Biology, Genentech, lnc., San Francisco, CA (U.S.A.)

(Received11 September1989) (Revised manuscript received2 February 1990)

Key words: Activin B; ActivinA; Protein purification; Growth factor Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the fl subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the fl subunit of inhibin B. Activin B has an apparent pl of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Introduction Activin is a member of the transforming growth factor-fl (TGF-fl) gene family [1,2]. Biologically, activin has been shown to stimulate both the secretion of follicle stimulating hormone (FSH) from anterior pituitary cells and the accumulation of hemoglobin in the K562 erythroid leukemic cell line [1,3]. Inhibin, another member of the TGF-fl gene family, shows biological effects opposite to those exhibited by activin in the systems described above [1,4]. Both inhibin [1,5,6] and activin [7,8] have been isolated from natural sources. Activin isolated from natural material is a covalent dimer exhibiting two forms, flAflA and flAflB" On the other hand, inhibin exhibits two covalent heterodimeric forms, aft A and aflB. The fl subunits are identical in

Abbreviations: TGF-fl, transforming growth factor-fl; FSH, follicle stimulating hormone; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morphohno)propanesulfonicacid; SSFF, S-Sepharose Fast Flow; NMWL, nominal molecular weight limit; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamidegel electrophoresis; Bistris, bis-(2-hydroxyethyl)iminotris(hydroxymethyl)methane; RP-HPLC, reversed-phasehigh-performanceliquid chromatography; IEF, isoelectric focusing; TCF, tissue culture fluid; UF/DF, ultrafiltered/diafiltered. Correspondence: C.H. Sehraelzer,Genentech, Inc., RecoveryProcess R&D, 460 Point San Bruno Blvd., South San Francisco, CA 94080, U.S.A.

both activin and inhibin. Recombinant human activin A (flAflA) has been isolated and shown to have biochemical properties and biological activities similar to its native counterpart isolated from ovarian follicular fluid [7-9]. To date, activin B (flBfla) has been postulated [2], but not yet isolated from natural sources. Although fib mRNA has been detected in the ovary, testis, placenta, pituitary, and whole brain [10], only recently has the fib m R N A been found alone in granulosa cells of small antral follicles [11]. Based on the nucleotide sequence of the c D N A encoding the fib subunit [12], recombinant activin B has recently been expressed in mammalian kidney cells [13]. We present here a method for the purification and some biochemical characterization of the recombinant activin B molecule purified from those cells. This is the first report describing the purification of this novel protein. The availability of purified activin B is crucial to the complete structural and functional characterization of the protein and provides an opportunity to increase our understanding of the biological role of this interesting protein. Experimental procedures Materials

S-Sepharose Fast Flow, phenyl-Sepharose CL-4B, Sephacryl S-300 HR, low-molecular-weight SDS standards, isoelectric focusing standards (3-10), PhastGel IEF 3-9, polybuffer 74, Mono P H R 5 / 2 0 and a Superose 12 H R 10/30 were purchased from Pharmacia LKB

0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

136 Biotechnology, Inc. YM10 ultrafiltration membranes were from Amicon. A microdialysis system was purchased from Bethesda Research Laboratories. The Vydac C a 214TP]010 reversed-phase H P L C column (1.0 z 25 cm) was obtained from P h a s e Separations Group. Pellicon cassette membranes (10000 N M W L , Cat. No. P T G C 000 05) were purchased from Millipore. Enhance was purchased from New England Nuclear. Acetonitrile was obtained from Baker Chemical Co. Trifluoroacetic acid was from Pierce. Bio-Lyte 3 / 1 0 carrier ampholytes were from Bio-Rad. Myoglobin (M9267), carbonic anhydrase (C6403), trypsinogen (Tl146), trypsin inhibitor (T1021), /3-1actoglobulin A (L5137), ovalbumin (A2512), cytochrome c (C7752), aprotinin (Al153), Blue dextran (D4772) and dithiothreitol were purchased from Sigma. Urea was from Mallinckrodt and was prepared as a stock 7.5 M solution and deionized using a mixed bed resin prior to use. All other chemicals were of reagent grade.

Methods Expression of activin B. Details of the expression of activin B are described elsewhere [13]. Briefly, a fulllength coding sequence for the fib subunit of inhibin was inserted into a cytomegalovirus expression vector which was used to transfect a mammalian kidney cell line [9,14]. Neomycin-resistant clones were selected and shown to secrete biologically competent activin B [13]. Purification of actioin B. 40 1 of tissue culture fluid (TCF) containing recombinant activin B from mammalian kidney cells were concentrated approx. 80-fold at 4 ° C and diafiltered into 25 m M Mes, 6 M urea (pH 6.5) using a pellicon tangential flow membrane (polysulfone) concentrator (10 square feet N M W L 10000). All chromatography steps were performed at room temperature unless otherwise indicated. The U F / D F concentrated T C F was applied to a 5 x 13 cm SSFF column equilibrated in 25 m M Mes, 6 M urea (pH 6.5) at a flow rate of 15 m l / m i n . The flow-through fraction, containing activin B, was adjusted to 0.8 M NaC1 and applied to a 2.5 x 9 cm phenyl Sepharose CL-4B column equilibrated in 25 m M Mops, 0.8 M NaCI, 6 M urea, (pH 7.0) at a flow rate of 5 m l / m i n . The column was washed with equilibration buffer (3 bed volumes) until the A280 returned to near baseline, followed by 9.5 bed volumes of 25 m M Mops, 6 M urea (pH 7.0). The column was eluted with a solution consisting of 40% ethanol and 60% 25 m M Mops, 6 M urea (pH 7.0). Activin B was pooled and concentrated approx. 10-fold in an Amicon stirred cell using a YM10 membrane. The concentrated pool was applied to a Sephacryl S-300 H R column (2.5 x 110 cm) equilibrated with 1 M acetic acid, 7.1 M urea at a flow rate of 0.8 m l / m i n at 4 ° C . The activin B was pooled and loaded directly onto a Vydac C 4 R P - H P L C (1.0 x 25 cm) column at 4 m l / m i n using a Waters 600 E H P L C system. Solvent A was

aqueous 0.1% trifluoroacetic acid (TFA) and solvent B was 0.1% T F A in acetonitrile. After loading of sample, the flow rate was reduced to 3 m l / m i n and the column was washed for 5 min with 32% B. Activin B was eluted using a linear gradient of 32 to 38% B in 75 min. Fractions containing activin B were pooled and stored at 4 ° C for biological and chemical characterization. SDS polyacrylamide gel electrophoresis. Slab gel electrophoresis was performed in a 12.5% resolving gel with a 4% stacking gel according to the method of Laemmli [15]. The gels were silver-stained according to Morrisey [161. Gel filtration. The molecular weight of activin B was determined by gel filtration on a Superose 12 column (1 x 30 cm) equilibrated in 25 m M Tris-HCl (pH 7.2) containing 6 M guanidine hydrochloride. The flow rate was 0.5 m l / m i n . Reduced activin samples (10 ~g) were prepared prior to chromatography by reduction at 80 o C for 5 min in equilibration buffer containing 100-fold molar excess of dithiothreitol over disulfide bonds. Other samples were dissolved in equilibration buffer at room temperature prior to chromatography. The Superose 12 column was calibrated using ovalbumin ( M r = 43 000), carbonic anhydrase ( M r = 30 000), cytochrome c ( M 12400) and aprotinin ( M r = 6500). Isoelectric focusing. The p I for activin B was determined using a Pharmacia Phast gel system (PhastGel IEF 3-9). Prior to use the I E F gel was soaked in Bio-Lyte 3 / 1 0 carrier ampholyte solution containing 6 M deionized urea. The p H gradient was determined using calibrated p I standards: (Broad p I calibration kit 3-10: myoglobin (6.76, 7.16) carbonic anhydrase (5.85), trypsin inhibitor (4.55), and trypsinogen (9.3)). Protein sequencing. The amino-terminal sequence of activin B was determined using an Applied Biosystem 477 sequenator with an on line 120A HPLC. Amino acid analysis. Amino acid compositions were determined using a Beckman 6300 amino acid analyzer. Samples were hydrolyzed for 24 h at l l 0 ° C in 6 M HC1 prior to analysis. Chromatofocusing. Chromatofocusing was performed using a Mono P H R 5 / 2 0 column. The column was equilibrated in 25 m M Bistris (pH 6) containing 6 M urea. The flow rate was 0.5 m l / m i n . Purified activin B was eluted from the Mono P column using 10% polybuffer 74 (pH 4.0) in 6 M urea. 1-ml fractions were collected. The p H gradient profile was determined using a Corning 125 p H meter. Extinction coefficient. The extinction coefficient for activin B was determined using a Hewlett Packard 8452A Diode Array spectrophotometer. The protein was dialyzed and the absorbance was determined in 0.05 M acetic acid. The concentration of activin B was determined by amino acid analysis. Protein assay. Protein concentration was determined by the method of Bradford [17] using the Pierce protein r

=

137 assay reagent. Activin B quantitated by amino acid analysis was used as the standard. Protein concentrations of purified samples were also determined either by amino acid analysis or by absorbance at 280 nm using 280nm of 1.8. 80.1% I cm Immunoprecipitations. Transfected or stable clones expressing either activin A or activin B were grown in the presence of [35S]methionine and [35S]cysteine [13]. For immunoprecipitation of activin A, protein A-purified monoclonal antibody prepared against recombinant activin A was used. For activin B, ammonium sulfateprecipitated goat polyclonal antibody raised against purified recombinant activin B was used. Recombinant activin A was immunoprecipitated from the labelled supernatants (50/~ 1) with 2/tg of affinity-purified activin A IgG at room temperature for 1 h. Recombinant activin B was immunoprecipitated from labeled supernatants (50 #1) with 2 #g of activin B IgG at room temperature for 1 h. Immune complexes were precipitated with protein A. Samples were separated on a 12% SDS-polyacrylamide gel, fixed in acetic acid, treated with Enhance, dried and analyzed by autoradiography. Bioassay. Activin B concentrations were determined by the stimulation of FSH release in cultured pituitary cells as previously described [9]. Activity is expressed as the concentration of activin B required to give a halfmaximal response in the assay (EDs0). 1 unit of activity is equivalent to 2.2 ng/ml of purified activin B. EDs0 values were calculated using a four-parameter curve-fitting program [18]. Results

Purification We describe here a simple, four-step purification which yields highly purified (more than 95%) activin B. The results of cation exchange chromatography of the concentrated tissue culture fluid on S-Sepharose fast flow are shown in Fig. 1. Activin B eluted in the flow

0.8-

c 0 e~

0.4-

g <

\ O1000 Volume

2000 (ml)

Fig. 1. S-Sepharose Fast Flow Chromatography. The concentrated tissue culture fluid in 25 mM Mes, 6 M urea, p H 6.5 was subjected to S-Sepharose chromatography as described under 'Experimental Procedures'. The flow through fractions containing activin B were pooled as indicated by the brackets further purification.

through fractions. While, substantial amounts (more than 90% by protein determinations) of the host contaminant proteins were removed (Table I and Fig. 5, lane 2). The flow through pool was adjusted to 0.8 M in NaC1 and chromatographed on a phenyl Sepharose CL-4B hydrophobic interaction column (Fig. 2). Activin B bound to the phenyl Sepharose column, while the majority of the remaining protein contaminants flowed through. Activin B eluted in two regions of the chro-

TABLE 1

Purification of activin B from tissue culture fluid Step

Volume (ml)

Protein (mg)

Total units (- 10-4)

Specific activity (U/mg) a (- 10- 3)

Purification (fold)

Recovery (%)

Tissue culture fluid concentrate SSFF FT Pbenyl Sepharose CL-4B Pool E S-300 HPLC

800 1360 56 45 24

7 740 610 6.7 2.8 b 1.6 d

1144.0 802.4 147.3 n.d. c 85.7

1.48 13.2 219.8 _ 535.5

1 8.9 149 _ 360

100 70 12.8 _ 7.5

a 1 U of activity is the amount of activin B required to give a half-maximal response in the pituitary bioassay. Typically, 1 U for purified activin B is 2.2 n g / m l [12]. b Protein determined by absorbance at 280 nm. c n.d., not determined. o Protein determined by amino acid composition.

138 0.4 Pool E 0.8c

c

o

0 cO

0.6Pool M

0.2c o .o

Q4-

m

,I<

<

02-

r i

750

150(

17150

I 0

2 0'0 0

Volume (ml

Volume

Fig. 2. Phenyl Sepharose CL-4B chromatography of activin B. The S-Sepharose flow-through pool was applied to the phenyl Sepharose column and eluted as described under Experimental procedures. The fraction eluting in the low conductivity eluant (25 m M Mops, 6 M urea (pH 7.0), elution volumes 1600-1900) is referred to as 'Pool M' and the ethanol-eluted material (elution volumes 1900-2000) is called 'Pool E' in the figure.

matogram; the low conductivity pool (pool M) and in the ethanol elution pool (pool E) (Fig. 5, lane 3). The greatest purity for activin B resulted when pool E alone was further purified. Pool E was U F / D F into 25 mM Mops, 6 M urea (pH 7.0) to remove ethanol prior to diafiltration into 1 M acetic acid 7.1 M urea. If ethanol was present during diafiltration into acid, activin B precipitated (data not shown). Concentrated/ diafiltered pool E was chromatographed on Sephacryl S-300 HR in 1 M acetic acid/7.1 M urea (Fig. 3). This removes most of the higher-molecular-weight contaminants, and yields an activin B pool which appears reasonably pure on SDS-PAGE (Fig. 5, lane 4). To remove some minor contaminating proteins, the activin B-containing fractions were pooled and loaded directly onto a Vydac butyl reversed-phase column. Most of the remaining host contaminants eluted in the leading edge

0.2

c 0 0O v 0.1

8c

\

<

I

0

I

200 Volume

I 30

400

600

(ml)

Fig. 3. Sephacryl S-300 chromatography of the phenyl Sepharose ethanol-eluted fractions (Pool E). Column fractions were analyzed by SDS-PAGE and the activin B containing fractions (indicated by brackets) were pooled as described under Experimental procedures.

I 60

I 90

I 120

(ml)

Fig. 4. Elution profile of activin B from reversed-phase HPLC. The activin B pool from the Sephacryl S-300 column was applied to a Vydac C 4 column and eluted as described under Experimental procedures. Fractions containing activin B were pooled as indicated by the brackets.

of the activin B peak (Fig. 4). Fractions containing activin B (determined by SDS-PAGE) were pooled and subsequently used for protein characterization. From 40 1 of tissue culture fluid, we recovered 1.6 mg of highly purified activin B (Table I). Characterization

The amino acid composition of activin B (Table II) is consistent with the theoretical composition expected from the /3 subunit of inhibin B [12]. Amino acid analysis yielded low isoleucine and valine amounts, attributable to one lie-lie bond and two Ile-Val bonds T A B L E I1 Amino acid composition of recombinant actioin B Residue

Asx Thr Ser Glx Pro Gly Ala Cys Val Met lie Leu Tyr Phe His Lys Arg Trp

Residues/monomer theoretical

observed

14 6 7 8 6 11 6 9 7 4 7 7 7 5 1 2 6 2

13.6 + 0.1 a 6.0 + 0.3 5.8 + 0.4 8.2 + 0.1 5.8 ± 0.6 11.0±0.2 6.0 ± 0.0 7.6 ± 0.2 6.3 ± 0.1 3.5 ± 0.1 5.7 + 0.1 7.0 ± 0.0 6.3 ± 0.2 5.0 ± 0.1 1.1 ± 0.1 2.1 + 0.1 5.8±0.1 n.d. b

" Based on 6 preparations of activin B, mean ± S.D. b n.d., not determined.

139 015

TABLE III

Amino-terminal sequence analysis of recombinant activin B J Cycle

Residue

Amount (pmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Gly Leu Gly Cys a Asp Gly Arg Thr Asn Leu Cys a Cys a Arg Gin Gin

216.5 209.3 139.5 _ 192.1 159.7 213.9 116.4 120.1 108.7 _ _ 106.8 66.6 93.3

2

3

4

5

0 - ~

j

-6

,-7 0,10-

I o I

0

-sz

o

tx

0.05-

-4

o

o

2'0

6

kDo

- 67

- 43

-- 3 0

I

,~ Volume

a No amino acid was found in this cycle, but Cys is expected from the sequence.

1

0--0

6o

,o

(ml)

Fig. 6. Chromatofocusing of purified activin B. Purified activin B was exchanged into 25 mM Bistris, 6 M urea (pH 6.0) and loaded onto a Mono P 5/20 column. Activin B was eluted using polybuffer 74 diluted 10-fold into 6 M urea and adjusted to pH 4.

in the activin B protein sequence. Amino-terminal sequence analysis (15 residues) of the purified activin B pool (Table III) was consistent with the expected amino terminus of the fl subunit of inhibin B deduced from the D N A sequence. Activin B gave a single band with a pI of 4.6 on isoelectric focusing (data not shown). Chromatofocusing on a Mono P column (HR5/20) gave a single peak at pH 4.7 (Fig. 6). The molecular weight of activin B determined by SDS-PAGE was 25900 unreduced (Fig. 5) and 15200 following reduction (data not shown). The molecular weight of the dimer as determined by SDS-PAGE agrees with the M r calculated from the amino acid composition

5.0

- 20.1

4.5

-14.4

..J

Fig. 5. SDS-polyacrylamide gel electrophoresis of activin B at various stages of purification. Samples were dialyzed overnight against 0.1 M acetic acid in a microdialysis system. The sample aliquots were dried under vacuum, prepared and subjected to 12.5% SDS-PAGE as described under Experimental procedures. Lane 1, concentrated tissue culture fluid; lane 2, S-Sepharose flow-through pool; lane 3, the ethanol-eluted pool from the phenyl Sepharose column; lane 4, Sephacryi S-300 column pool; Lane 5, RP-HPLC activin B pool; lane 6, molecular weight standards, bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa).

4.0

3.5 0.2

i

I

i

0.30

0.40

0.50

Kov Fig. 7. Analytical gel filtration of activin B. Aetivin B (A) and standard proteins (O) were chromatographed on a Superose 12 (1.0 x 30 cm) column as described under Experimental procedures.

140 2

3 kDo -92.5 -69 -46 -30

-18.4 -14.3

Fig. 8. Immunoprecipitations of activin A and activin B. Radiolabeled supernatants containing activin A or activin B were immunoprecipitated using either a monoclonal antibody raised against recombinant activin A or a polyclonal antibody prepared against activin B. Immunoprecipitation using activin A antibody: lane 1, activin A; lane 2, activin B; and immunoprecipitation using activin B polyclonal antibody: lane 3, activin B; lane 4, activin A.

(25 600). Surprisingly the molecular weight of activin B, as determined by gel filtration in 6 M guanidine hydrochloride was approximately 11000 whether the protein was reduced or not (Fig. 7). This suggests dimeric activin B retains a very compact structure even in this strongly denaturing solvent. The ~max for activin B is 278 nm. The extinction coefficient at 1 m g / m l and 1 cm light path at 280 nm is 1.8 in 0.05 M acetic acid. A monoclonal antibody prepared against recombinant activin A shows little or no cross reactivity with recombinant activity B (Fig. 8). A goat polyclonal antibody prepared against activin B, also shows virtually no cross reactivity with activin A (Fig. 8). Discussion

This is the first report detailing the purification of human recombinant activin B, a molecule not yet isolated from a natural source. We have isolated recombinant activin B to more than 95% purity, with an overall yield of 7.5%. A typical preparation provided 1.6 mg of highly purified activin B from 40 l of tissue culture culture fluid. The key steps in the purification procedure are the S-Sepharose fast flow and phenyl Sepharose CL-4B columns, which produce a total purification of 149-fold. Purified activin B had the expected N-terminal sequence and amino acid composition as deduced from the cDNA coding for the fl subunit of inhibin B [12]. Despite the close similarity (65% identical) between activin B and activin A, they show a wide difference in isoelectric points; for activin B the p I is 4.6 and for

activin A the p I is 8.6 (Schmelzer, C.H. and Burton, L.E., unpublished data). Activin B exhibits a very compact structure, eluting from the sizing column (in 6 M guanidine hydrochloride) with an apparent molecular weight of about 11 000. We have found the same result for activin A (Schmelzer, C.H. and Burton, L.E., unpublished data). The present work shows that activin B is quite stable in buffers containing urea a n d / o r 1 M acetic acid. Previous studies have shown that activin A retains activity in some denaturants such as 6 M guanidine hydrochloride [7], buffers containing 6 M urea, 30% acetic acid [8] and following SDS-PAGE [19]. The involvement of specific intra a n d / o r intermolecular disulfide bonds in biological activity has not been determined. However, previous studies have shown that the native dimer is essential for biological activity, since reduction of disulfide bonds causes complete loss of activity [7,9]. Activin B and activin A exhibit similar activities and biological potencies in both the rat pituitary FSH release assay and the K562 differentiation assay [13]. Despite their similarities in sequence, size and biological activities, these molecules have regions which differ as shown by antibodies which are specific to each activin. The lack of antibody cross reactivity could be related to apparent hydrophilic differences expressed by the overall difference in the isoelectric points of the two proteins. Several non-identical regions exists between these two molecules, e.g., residues 19-22 and 71-78, reflecting some of these hydrophilic differences. In addition, the interspecies similarities and the high level of homogeneity between activins A and B could result in a limited number of epitopes available for polyclonal antibody production. This may explain the lack of cross reactivity with activin A by the activin B goat polyclonal antibody. The amino acid sequence conservation of activin A (95-100%) across species and the conservation of the fib subunits among human, rat and porcine sequences (95100%) [2,5,12] strongly suggests that the 25-35% sequence variation between the activins could be important for some as yet undiscovered functional differences between them. As previously mentioned, both the homodimeric activin A and the heterodimeric activin AB have been isolated from natural tissues [7,8] while homodimeric activin B has not. Until recently, whenever fib m R N A was detected in a variety of tissues, it was found colocalized with a m R N A [10]. The discovery that only fib m R N A was found in granulosa ce',ls of small antral follicles offers an opportunity to look for the activin B molecule in a natural source [11]. Monoclonal activin B antibodies may prove very useful for searching in other tissues for activin B and for isolating small quantities of activin B from natural sources such as the small antral follicles.

141

The availability of recombinant sources of the TGF-fl family of proteins will be instrumental in the study of structure-function relationships within this group of related proteins. Recent efforts have produced recombinant TGF-fl 1 [20], activin A [9], activin B [13] and inhibin (Mason, A.J., personal communication). Primary structure comparisons, using cDNA or natural protein sequences reveal the potential for a common disulfide bridging structure in these proteins, which remains to be elucidated. Analogous to the insulin family of proteins [21], the primary structural differences of the TGF-fl family of proteins will likely lead to an interest in the relationship of the biological activities of its members and their possible receptor cross reactivities [22]. In summary, we have isolated recombinant human activin B using a rapid, simple purification scheme. The molecule isolated reflects the predicted primary structure determined by the cDNA of the fl subunit of inhibin B and stimulates FSH secretion in pituitary cells with a potency comparable to natural and recombinant human activin A. Additionally, recombinant human activin B, like activin A, has been shown to stimulate hemoglobin production in the K562 assay system. References 1 Mason, A.J., Hayrick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S.-Y., Guillemin, R., Niali, H., Seeburg, P.H. (1985) Nature 318, 659-663. 2 Ying, S.-Y. (1987) Proc. Soc. Exp. Biol. Med. 186, 253-264. 3 Yu, J., Shao, L., Lemas, V., Yu, A.L., Vaughan, J., Rivier, J. and Vale, W. (1987) Nature 330, 765-767.

4 Rivier, J., Spiess, J., McClintock, R., Vaughan, J., Vale, W. (1985) Biochem. Biophys. Res. Commun. 133, 120-127. 5 Ling, N., Ying, S.-Y., Ueno, N., Esch, F., Denoroy, L. and Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82, 7217-7221. 6 DeJong, F.H. (1988) Physiol. Rev. 68, 555-607. 7 Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Kan, D. and Speiss, J. (1986) Nature 321, 776-779. 8 Ling, N., Ying, S.-Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, M. and Guillermin, R. (1986) Nature 321,779-782. 9 Schwall, B,H., Nikolics, K., Szonyi, E., Gorman, C. and Mason, A.J. (1988) Mol. Endocrinol. 2, 1237-1242. 10 Meunier, H., Rivier, C., Evans, R.M. and Vale, W. (1988) Proc. Natl. Acad. Sci. USA 85, 247-251. 11 Schwall, R.H., Mason, A.J., Wilcox, J.N., Bassett, S.G. and Zeleznick, A.J. (1990) Mol. Endocrinol., in press. 12 Mason, A.J., Niall, H.D. and Seeburg, P.H. (1986) Biochem. Biophys. Res. Commun. 135, 957-964. 13 Mason, A.J., Berkemeier, L.M., Schmelzer, C.H. and Schwall, R.H. (1989) Mol. Endrocrinol. 3, 1352-1358. 14 Eaton, D.L., Wood, W.I., Eaton, D., Hass, P.E., Hollingshead, P., Wion, K., Mather, J., Lawn, R.M., Vehar, G.A., Gorman, C. (1986) Biochemistry 25, 8343-8347. 15 Laemmli, U. (1970) Nature 227, 680-685. 16 Morrisey, J.H. (1981) Anal. Biochem. 117, 307-310. 17 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 18 Marquardt, D. (1963) J. Soc. Indust. Appl. Math. 11,431-443. 19 Ling, N., Ying, S.-Y., Ueno, N., Shimsaki, S., Esch, F., Hotta, M. and Guillemin, R. (1986) Biochem. Biophys. Res. Commun. 138, 1129-1137. 20 Derynck, R., Jarrett, J.A., Chen, E.Y., Eaton, D.H., Bell, J.R., Assoian, R.K., Roberts, A.B., Sporn, M.B. and Goeddel, D.V. (1985) Nature 316, 701-705. 21 Blundell, T.L. and Humbel, R.E. (1980) Nature 287, 781-787. 22 Campen, C.A. and Vale, W. (1988) Biochem. Biophys. Res. Commun. 157, 844-849.