Amino/Carboxyl-terminal Deletion Mutants of Human ...

Vnl. 268. No. 13, Issue of May 5,pp. 9311-9315,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Amino/Carboxyl-terminal Deletion Mutants of Human Choriogonadotropin ,8* (Received for publication, November 4, 1992)

Jianing HuangS, Fang Chens, and David PuettSll From the Reproductive Sciences and Endocrinology Laboratories, Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101

to receptor binding or to interfere with FSH receptor or TSH receptor binding. Consistent with this possibility were the findings that synthetic peptides based on amino acid residues 1-16 of hCGP and onresidues 1-15 of the P subunit of human LH and CG inhibited binding of intact hCG to the CG/LH receptor (Salesse et al., 1990; Keutmann et al., 1991). The placental CGPs contain 145-149 amino acid residues and also have a glycosylated carboxyl-terminal extension of over 20 amino acid residues as does equine LHP (Ward et al., 1991). We and othershave used site-directed mutagenesis to introduce premature termination codons and have shownthat this carboxyl-terminal extension in hCGp is not required for et al., 1989; Matzuk receptor binding and activation (El-Deiry et al., 1990; Chen and Puett, 1991a; Chen and Bahl, 1991), although it does appear to increase the circulatory half-life of hCG (Matzuk et al., 1990). We investigated the contribution of other carboxyl-terminal amino acid residues in hCGp by preparing and characterizing des( 101-145)hCGP and des(93145)hCGP (Chen and Puett,1991a). Interestingly, the former bound to cy and was bioactive i n vitro, albeit with reduced potency relative to thewild type hormone; in contrast, the192 fragment failed to associate with a. These results defined 1-100 as the shortest amino-terminal fragmentof hCGp that could form an active holoprotein. Earlier studies have demonstrated thatdes(122-145)hCGP (El-Deiry et al., 1989) and des(115-145)hCGP (Matzuk et al., 1990; Chen and Bahl, 1991) exhibit properties i n vitro indistinguishable from hCGp wild type, including a binding, recepThe glycoprotein hormones are heterodimers consistingof tor binding, and receptor activation, while des(lO1-145)hCGP a common a subunit anda hormone-specific P subunit (Ryan is reduced in potency (Chen and Puett, 1991a). We prepared et al., 1988). The P subunits are homologous, but there are intermediate an carboxyl-terminal deletion fragment, interesting amino acid differences in the chain lengths. For des(lll-l45)hCGP, to localize the region in the amino acid example, the lutropin and choriogonadotropin subunits sequence, where i n vitro potency begins to decrease relative contain an extension of 6 or 7 amino acidresidues at the to hCGp wild type. T o investigate the contribution of the amino terminus that are not present in follitropin and thy- amino-terminalextension,thedeletionfragment, des( 1rotropin (Ward et al., 1991). Since LH1 andCG act through a 7)hCGP, wasprepared and characterized. In continuing efforts common G protein-coupled receptor (Segaloff et al., 1990), to identify the shortest core structure of hCGP required for the amino-terminal extension may contribute in some mannerproper folding, a binding, receptorbinding, and receptor activation, two combined amino-terminal and carboxyl-ter* This research was supported by Research Grant DK33973 from the National Institutes of Health. The costs of publication of this minal deletions were prepared and characterized, des( 1-7, article were defrayed in part by the payment of page charges. This 111-145)hCGP and des(1-7, 101-145)hCGP. Our results inarticle must therefore be hereby marked “advertisement” in accord- dicate that hCGp fragment8-110 can form an active heteroance with 18 U.S.C. Section 1734 solely to indicate this fact. dimer, while the 8-100 fragment does not bind with high 4 Present address: Dept. of Biochemistry, The University of Geor- affinity to cy, perhaps because of an inability tofold properly. gia, Athens, GA 30602.

Human choriogonadotropin (hCG)contains an a subunit common to other membersof the glycoprotein hormone family, lutropin (LH), follitropin, and thyrotropin, and a hormone-specific B subunit. hCGB contains a carboxyl-terminal extension of 25-30 amino acid residues not present in the other B subunits; also, CGB and lutropin B have an additional 6 or 7 aminoterminal residues that are not present in follitropin B and thyrotropin 8. To delineate the contribution of these extensions in hCGB, site-directed mutagenesis was used to prepare several deletion fragments. Plasmids containing cDNAs for wild-type and mutant hCGB were transiently transfected into Chinese hamster ovarycells containing a stably integrated gene for bovine a. Medium from the transfected cells was used in twoin vitro assays witha transformed murine Leydig cell line, MA-10. The deletion fragments, des(17), des(111-145), and des(1-7, 111-145), associated with a as well as hCGB wild-type; moreover, the potencies of the three mutant hormones were comparable to that of control. In contrast, des(1-7, 101-145)hCGB yielded very little heterodimer, although that which formed was partially active. These results define the shortest known core fragment of hCGj3, amino acid residues 8-1 10, that retains significant functionality in vitro.

Present address: Dept. of Cell Biology, Baylor College of Medicine, Houston, T X 77006. ll To whom reprint requests should be addressed Dept. of Biochemistry, Life Sciences Building, The University of Georgia, Athens, GA 30602. The abbreviations used are: LH, lutropin; CG, choriogonadotropin; hCG, human choriogonadotropin; EDSnreffective dose for 50% inhibition of binding or 50% stimulation of progesterone relative to control; FSH, follitropin; and TSH, thyrotropin; PCR, polymerase chain reaction.

EXPERIMENTALPROCEDURES

Materials-The site-directed mutagenesis kit (Muta-gene In Vitro mutagenesis kit) was from Bio-Rad, andthe expression vectors pcDNAI/NEO and pRSV were obtained from Invitrogen (San Diego, CA) and Dr. John H. Nilson (Case-Western Reserve University, Cleveland, OH), respectively. The Sequenase version 2 kit was a product of United States Biochemical Corp. Centricon 10 columns were from Amicon, and plasmid purification columns were from Qiagen (Chatsworth, CA). W~~S-CIATP 500 (Ci/mmol) and [1,2,6,7-

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hCGp Deletion Mutants

3H]progesterone (94 Ci/mmol) were from Du Pont-New England Nuclear; progesterone and a progesterone antiserum were purchased from Steraloids (Wilton, NH) and Radioassay Systems Laboratories (Carson, CA), respectively. A polyclonal antiserum directed against hCGp and 1251-labeled hCG (100-150 pCi/pg) were products of ICN (Horsham, PA), and the TANDEM-RHCG radioimmunoassay kit used to measure heterodimer concentrations was from Hybritech (San Diego,CA). Other reagents and supplies were obtained as given elsewhere (Chen and Puett, 1991a, 1991b). Preparation of Mutant and Wild-type hCGp cDNAs-The cDNA for hCGp was kindly provided by Dr. John Fiddes (California Biotechnology, Inc., Mountain View, CA) and inserted into the unique HindIII site of M13 mp18 (El-Deiry et al., 1989). To prepare the amino-terminal deletion mutant, des(l-7)hCGp, the cDNA was subcloned into the pRSV vector which served as template DNA for PCR mutagenesis. The following deoxyoligonucleotides were synthesized by Dr. Rudolf Werner (University of Miami) on an Applied Biosystems Model 380B DNA synthesizer. The four primers correspond to the following hCGp amino acid residues: (1)-2, -1, 8 to 14; (2) 9, 8, -1 to -7; (3) 10 to 15; and (4)-3 to -8. Primers 1 and 2 have an overlap region of -2 to -1 and of 8 to 9, which is used for annealing. Primer 1 : 5'-TGGGCACGGTGCCGCCCCATCAATGCC-3'(sense) Primer 2 : 5'-GCACCGTGCCCATGTCCCGCCCATGCT-3' ( a n t i s e n s e ) Primer 3 : 5'-CGCCCCATCAATGCCACC-3'(sense) Primer 4 : 5'-TGTCCCGCCCATGCTCAG-3' ( a n t i s e n s e )

for gonadotropin-enhanced progesterone production (Ascoli, 1981). The cells were kindly provided by Dr. Mario Ascoli (University of Iowa,Iowa City, IA) and grown as described elsewhere (Chen and Puett, 1991a, 1991b). Various amounts of expression medium containing known concentrations of the heterologous heterodimer, bovine a-hCGp (wild-type or mutant), were added to MA-10 cells in Waymouth's medium containing 1 mg of bovine serum albumin/ml. The competitive binding assay used 10' cpm of 1251-labeled hCG with a 16-h incubation at room temperature; nonspecific binding was determined in the presence of 2 pg of hCG. The cells were washed twice, trypsinized, and counted in a y-counter to determine bound radioactivity. The specific binding of 1251-labeled hCG was normalized to 100% in the absence of added expressed hormone, and the inhibition data are given relative to thisvalue. Progesterone was measured by radioimmunoassay in the cell medium following a 4-h incubation at 37 "C. Each assay contained bovine a-hCGPwild-type as a positive control, and reproducibility between assays, performed at least twice, was excellent. The progesterone data were normalized to 0% (basal) and 100% (maximal), the latter referring to the amount produced in response to 10 ng/ml of bovine a-hCGp wild-type, which was present in each assay. Generally basal progesterone production was between 3 and 5 ng/ml, and maximal production was in the range of 600-900 ng/ml. All results are presented as mean ? S.E. for a typical assay. RESULTS

DNA sequencing confirmed the presence of a start codon

at the appropriate position to ensure the absence of amino acid residues 1-7 in the des(1-7)hCGp mutant and t h e locations of the premature termination codonsas expected in the carboxyl-terminal and combinedamino/carboxyl-terminal SEQUENCES 1-4 deletion mutants. Sequencing also verifiedthat no other mutations had occurred in the wild-type and mutant cDNAs. Mutagenesis was performed by PCR (Jones and Howard, 1990) The amino-terminal deletion mutant, des(l-7)hCGP, and with primers 1 and 4 and primers 2 and 3 separately. The PCRamplified plasmids were purified on a 1%agarose gel; the two deletion the carboxyl-terminal deletion mutant, des(lll-l45)hCG& plasmids were annealed, and transformation of the nicked plasmid were expressed at about the same level as hCGp wild-type into DH5a cells was done via electrotransformation. HindIII frag- and each combined with bovineCY as well as hCGp wild-type ments from a number of clones were subcloned into M13 mp18 for under the conditions used. For example, the percentage of sequencing (Sanger et al., 1977); several clones were identified which total expressed hCGp in heterodimer form was in the range had the expected sequence of the wild-type cDNA for hCGp with the and for hCGp wild-type. T h e exception of the desired deletion. The mutant gene was subcloned of 5 0 4 0 % for the mutants des(1-7, back to pcDNAI/NEO and to pRSV; the plasmids with the correct combined amino/carboxyl-terminal deletion mutant, lll-l45)hCGP, was also expressedat about the samelevel as orientation were amplified (Maniatiset al., 1982), and DNAwas prepared. hCGp wild-type, and the percentageof total expressed mutant Carboxyl-terminal deletion mutants of hCGp were prepared using P forming holoprotein was only slightly less than that obwild-type cDNA and the mutant cDNA for des(l-7)hCGP described tained with hCGp wild-type. T h e a m o u n t of des(1-7, 101above. These were designed to yield the carboxyl-terminal deletion mutant, des(lll-l45)hCGP, and the combined amino/carboxyl-ter- 145)hCGP measured by the radioimmunoassay was only about minal deletion mutants, des(1-7, 101-145)hCGP and des(1-7, 111- 25% that of hCGp wild-type, and only 1-2% of the measured 145)hCGP. For these, the desired 21-base deoxyoligonucleotidescon- m u t a n t P formed heterodimer. taining the appropriate premature termination codon were syntheThe holoprotein formed betweenCY and des(1-7)hCGP was sized as described above. Mutagenesis was performed with the Muta- active in receptor binding and activation (Fig. l),although gene In Vitro mutagenesis kit, and mutant phage clones were identi- the potency, as estimated bythe ED50 for inhibitionof binding fied by sequencing. The desired mutant genes were subcloned into a pRSV expression vector, obtained by self-ligation of the one contain- or stimulation of progesterone, was somewhat reduced relative wild-type. The holoprotein formed between th ing the hCGp wild-type gene, and plasmids were amplified and DNA t o t h a t w i hCGP CY and des(lll-145)hCGPwas,withinexperimentalerror, prepared. Expression and Measurement of Wild-type and Mutant hCGpsthat obtained withhCG@wildequipotent in both assays with The wild-type and deletion mutants were each transfected into CHOa type (Fig. 2). With both mutant gonadotropins the maximal cells via the dimethyl sulfoxide/Polybrene method (Chaney et al., a m o u n t of progesterone production, e.g. as evaluated at a 1986). These cells, kindly provided by Dr. John H. Nilson (CaseWestern Reserve University, Cleveland, OH), contain a stably inte- gonadotropin concentration of 10 ng/ml, was equivalent to grated gene for bovine a (Kaetzel et al., 1985). Medium from the that in response tothe control, cY-hCGp wild-type. The combined amino/carboxyl-terminal deletion mutant, transfected cells, which were maintained as described elsewhere (ElDeiry et al., 1989; Chen and Puett, 1991a, 1991b), was collected about des(1-7, lll-l45)hCGP, yielded a heterodimer that was also 1 week after transfection and concentrated using Centricon 10 col- active and exhibited about the same potencies, i.e. ED50 valumns if desired. ues, in both assays as the standard a-hCGP wild-type (Fig. A specific polyclonal anti-hCGP antiserum, equally reactive with free p and the a6 complex, and lZ5I-hCGwere used to measure the 3). Also, the maximal amount of progesterone produced in concentration of total hCGp in the cell medium. A solid phase, two- response to this mutant gonadotropin was equivalentto that to wild-type. The small amount site immunoradiometric assay was used to determine the concentra- produced in response a-hCGP tion of heterodimer in the medium. In this assay, a mouse monoclonal of heterodimer that formed with des(1-7, 101-145)hCGP was antibody directed against an epitope on hCGa and an lZ51-labeled active, butless so than a-hCGP wild-type. After concentrating mouse monoclonal antibody against an epitope on hCGp in the a6 the expression medium, two independent steroidogenic assays complex were used.Radioactivity in bothassays was determined with at 0.15 ng/ml demonstratedthat the mutant gonadotropin,CYa y-counter (LKB Instruments). In Vitro Bioassays-The transformed murine Leydig cellline, MA- des( 1-7, 101-145)hCGP, stimulated progesterone production 10, was used for competitive binding assays with lzSI-labeledhCG and over basal, but to a much lesser degree than a-hCGP wild-

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hCGP Deletion Mutants 100

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FIG. 1. In vitro bioassays with bovine a-hCGj3 wild-type and bovine a-des( 1-7) hCGj3. A, competitive binding to MA-10 cells with lZ5I-labeledhCG and bovine a associated with hCG@wildtype (0)or des(l-7)hCGP (0).The specific binding of lZ5I-labeled hCG is normalized to 100% in the absence of expressed gonadotropin. B, stimulation of progesterone production in MA-10 cells by heterodimerscontaining bovine CY bound to hCG@ wild-type (0)or des(17)hCGP (0).The maximal amount of progesterone produced by bovine CYhCG@ wild-type was normalized to 100%.

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ir-Hormone Concentration(ng/ml) Within this CG/LH p amino-terminal extension, one tetrapeptide sequence corresponding to amino acid residues 4-7, Pro-Leu-Arg-Pro, is invariant in the 12 known mammalian CG/LH /3 sequences (Wardet aL, 1991). There appears tobe DISCUSSION a slight decrease in potency of the a-des(l-7)hCGp complex This study has demonstrated that the conserved 6- or 7- relative to a-hCGB wild-type, but small differences may not amino acid residue amino-terminal extension, present in the be significant since the radioimmunoassays used to measure p subunit of CG/LH but absent in thoseof FSH andTSH, is heterodimer concentrationsmay be influenced by the mutant in vitro. forms of p. In any case, a slightly reduced receptor affinity is not required for receptorbindingandactivation

type (Fig. 4). Due to the limited quantity of material available, a relative potency could not be estimated for this particular mutant gonadotropin.

hCGP Deletion Mutants

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FIG. 2. In vitro bioassays with bovine a-hCGO wild-type FIG. 3. In vitro bioassays withbovine cr-des(1-7, 111and bovine a-des(ll1-145)hCGB. A, see legend to Fig. 1A; displacement of lZ5I-labeledhCG by bovine a-des(ll1-145) hCGp (0). 145)hCGfl.A, see legendto Fig. l A ; displacement by bovine a-des(1B, see legend to Fig. 1B; stimulation by bovine a-des(ll1-145)hCGp 7, lllL145)hCGp (0).B , see legend to Fig. 1B; stimulation by bovine ~-des(l"i.,lll-l45)hCGp(0). (0).

not surprising since a synthetic peptide based on the amino interesting to determine whether thisregion of the CG/LH fi acid sequenceof hCGp 1-16 inhibited CY$ binding and binding subunit interferes with binding ofCG and LH to the FSH of '*'I-hCG to the CG/LH receptor on Leydig cells (Salesse and TSHreceptors. Other studieshave shown that heterodimers containing the et al., 1990). Also, synthetic peptidesbased on the aminoacid sequences of hCGP and hLHp 1-15 are capable of inhibiting carboxyl-terminal deletion mutants, des(115-145)hCGP binding of lZ5I-hCGto the CG/LH receptor, while replacement (Matzuk et al., 1990; ChenandBahl, 1991) and des(122of Lys-2 with Ala in these peptides abolished this inhibition 145)hCGP (El-Deiry et al., 1989), were equipotent i n vitro to (Keutmann et al., 1991). We found, using site-directed muta- that containinghCGP wild-type. However, a heterodimer with genesis, that replacement of Lys2 in hCGP with Glu did not des(101-145)hCGp, although active in uitro, exhibited greatly affect CY binding, but receptor binding and steroidogenic po- reduced receptor binding (Chen and Puett, 1991a). Our presaheterodimerwith des(ll1tency were reduced using i n uitro assays (Xia et al., 1993). entdatademonstratethat with one containinghCGp Thus, while the hCG@amino-terminal extension apparently 145)hCGP is essentially equipotent contributes to CG/LH receptor recognition, it is notrequired wild-type, implying that 1 or more amino acid residues befor high affinity binding and receptor activation. It will be tween positions 101-110 of hCGp contribute tohCG-receptor

hCGB Deletion Mutants

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be emphasized that FSHP and TSHP are devoid of the 1-7 region, yet they fold and bind to the same a subunit. An alternate explanation is that either amino acid residues 1-7 or 101-110 are required, either directly or indirectly, for a binding. A perusal of the aminoacid sequence of hCGP shows 15 the following regions of homology between amino acid residues 2-5 and 104-108: Lys-Glu-Pro-Leu and Lys-Asp-HisPro-Leu, respectively. These reduce to a (+)(-)/Pro-Leu sequence, where the positively and negatively charged residues arise from Lys and eitherAsp or Glu, respectively, and where the (+)(-) sequence is either adjacent to a Pro-Leu sequence or separated by His. The importance of this pseudo-internal repeat in the CG/LH p subunits, if any, remains to be demonstrated. The use of a heterologous gonadotropin in this study, i.e. bovine a-hCGP, is not expected to have any effect on the conclusions drawn regarding the role of specific amino acid residues of hCGP on function. In all cases the potencies of the gonadotropins containing hCGp mutantswere compared tothatwith hCGPwild-type,bovine a beingacommon a ‘b C subunit in each case. Moreover, we showed earlier that the FIG. 4. Steroidogenic assay using bovine a-des(1-7, 101146)hCGB. Progesterone production was measured in response to hybrid gonadotropin, ovine a-hCGP, exhibited receptor bindbovine a-hCGB wild-type ( a ) and tobovine cu-des(l-7,101-145)hCGP ing and activation properties in vitro like those of hCG and concluded that hCGP is predominant in determining activity, ( b ) ,each at 0.15 ng/ml; basal production is also indicated (c). while the natureof the a subunit serves more in a modulatory role (Strickland and Puett, 1981). The bovine and ovine a binding. Cys occurs at position 110 in hCGP and has been subunits have identical amino acid sequences, and thus we assigned to form a disulfidelinkage with CysZ6(Mise and Bahl, 1981). Huth et at. (1992) reported that this is the last expect the sameconclusion to hold for the heterologous hormone, bovine cu-hCGP. disulfide to form in hCGP, and its formation seems to be In summary, this study has demonstrated that the aminofavored by subunit association. It is quite conceivable that the C y ~ ~ ~ - C disulfide y s ” ~ stabilizes the@ subunit and, in turn, terminal7-amino acidresidue extension of hCGP can be the holoprotein and holoprotein-receptor complex. We have removed, as can 35 amino acid residues from the C terminus, shown that replacement of Lyslo4with Glu does not interfere without adversely affecting a binding or subsequent CG/LH with subunitassembly, but receptor binding decreased is (Xia receptor binding and activation by the holoprotein. Furtherfragment of hCGpconsisting of amino acid et al., 1993). It is noteworthy that position 104 is either Lys more,acore residues 8-110 was found to retain functionality, whereas that or Arg, i.e. apositively chargedamino acidresidue, inall known mammalian CG/LH p sequences; Gly’’’ is invariant of residues 8-100 only weakly associated with01. inthe known mammalianCG/LHamino acidsequences; REFERENCES and Pro andLeu at positions 107 and 108, respectively, occur Ascoli, M. (1981) Endocrinology 108,88-95 in 11/12 mammaliansequences(Ward et al., 1991). Thus, Campbell, R. K., Dean-Ernig, D. M., and Moyle, W. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,760-764 there are many possible candidates in this region to account Chaney, W.C., Howard, D. R., Pollard, J. W., Sallustio, S., and Stanley, P. (1986) Somat. Cell. Mol. Genet. 12, 237-244 for the differencein receptorbinding betweena mutant Chen, W., and Bahl, 0. P. (1991) J Biol. Chem. 266,6246-6251 gonadotropin containing the amino-terminal fragment1-100 Chen, F., and Puett, D. (1991a) J. B i d . Chem. 266,6904-6908 and one with 1-110. Interestingly, thisregion of the @ subunit Chen, F., and Puett, D. (1991b) Biochemistry 30, 10171-10175 El-Deiry, S., Kaetzel, D., Kennedy, G., Nilson, J., and Puett, D. (1989) Mol. hasbeen shown tobeimportantindeterminingreceptor Endocrinol. 3, 1523-1528 Huth, J. R., Mountjoy, K., Perini, F., and Ruddon, R. W. (1992) J. Biol. Chem. specificity (Campbell et al., 1991). 267,8870-8879 The data on the combined amino- and carboxyl-terminal Jones, D. H., and Howard, B. H. (1990) BioTechniqws 8 , 178-183 deletion mutantsof hCGP were intriguing and to some extentKaetzel, D. M., Browne, J. K., Wondisford, F., Nett, T. M., Thomason, A. R., and Nilson, J. H. (1985) Proc. Natl. Acad. Sci. U, S. A. 82, 7280-7283 surprising. That des(1-7, 111-145)hCGP bound to a , albeit Keutmann, H. T., Mason,K. A., Ruhin, D. A,, Kitzrnan, K., and Zschunke, M. (1991) Abstracts of the 73rd Annual Endocrine Society Meeting,p. 281 with an apparent affinity somewhat less than hCGP wildManiatis, T.,Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning:A type, toyield an active heterodimer with comparable potency Laboratory Manual, pp. 122-127 and 322-326, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY to a-hCGPwild-type is not totally unexpected since single the M. M., Hsueh, A. J. W., Lapolt, P., Tsafriri, A,, Keene, J. L., and deletions did not alter subunitassembly, receptor binding, or Matzuk, Boirne I. (1990) Endocrinology 1 2 6 , 376-383 receptor activation. The sequence 8-110 is the shortest frag- Mise, T., and Bahk, 0. P. (1981) J. Biol. Chern. 256, 6587-6592 R. J., Charlesworth, M. C., McCormick, D. J., Milius, R. P., and ment of hCGPknown to possess essentially full in vitro Ryan, Keutmann, H. T. (1988) FASEB J. 2, 2661-2669 activity. In contrast, our finding thatdes(1-7, 101-145)hCGp Salesse, R., Bidart, J.-M., Troalen, F., Bellet, F., and Garnier, J . (1990) Mol. Cell. Endocrinol. 68, 113-119 only weakly binds to a, while either single deletion appears Segaloff, D. L., Sprengel, R., Nikolics, K., and Ascoli, M. (1990) Rec. Progr. Harm. Res. 46,261-303 to bind to the a subunit in a comparable manner to hCGp Strickland, T. W., and Puett, D. (1981) Endocrinology 109, 1933-1942 wild-type (this work; ChenandPuett,1991a),impliesan Ward, D.,N., Bousfield, G. R., and Moore, K. H. (1991) in Reproduction in Domestrc Anmals (Cupps, P. T., ed) pp. 25-80, Academic Press, New York important stabilizingor 01 binding role foramino acid residues Xia, H., Huang, J., Chen, T.-M., and Puett, D. (1993) J. Mol. Endocrinol., in 1-7 and/or 101-110. While this is not unreasonable, it must press

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