Proc. Natl. Acad. Sci. USA Vol. 88, pp. 902-905, February 1991 Medical Sciences Thyrotropin-luteinizing /chorionic extracellular domain chimeras as probes for function (hormone binding/signal transduction) Yuji NAGAYAMA, HARRY L. WADSWORTH, GREGORIO D. CHAZENBALK, DIEGO Russo, Pui SETO, AND BASIL RAPOPORT Thyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, CA 94121; and the University of California, San Francisco, CA 94143-0534 Communicated by William J. Rutter, November 2, 1990

ABSTRACT To define the sites in the extracellular domain site(s) involved in ligand binding or the region(s) important in of the human thyrotropin (TSH) receptor that are involved in signal transduction. TSH binding and signal transduction we constructed chimeric Recent studies indicate that ligand- and antibody-binding thyrotropin-/chorionic gonadotropin sites in folded globular proteins are conformational and may (TSH-LH/CG) receptors. The extracellular domain of the consist of discontinuous regions of the linear amino acid human TSH receptor was divided into five regions that were sequence (13-17). In studies defining ligand-binding sites in replaced, either singly or in various combinations, with ho- native proteins it is therefore important to conserve the mologous regions of the rat LH/CG receptor. The chimeric three-dimensional structure of the protein. The considerable receptors were stably expressed in Chinese hamster ovary cells. (30-50%) homology in the extracellular domains of the gly- The data obtained suggest that the carboxyl region of the coprotein hormone receptors together with the presence of 10 extracellular domain (amino acid residues 261418) and par- conserved extracellular cysteine residues [some ofwhich are ticularly the middle region (residues 171-260) play a role in thought to form disulfide bonds (12)] suggest a similar three- signal transduction. The possibility is also raised of an inter- dimensional structure for the extracellular domains of these action between the amino and carboxyl regions of the extra- receptors and make them ideal candidates for homologous cellular domain in the process of signal transduction. With substitution studies of these regions. For this purpose we respect to hormone binding, substitution of the entire extra- performed homologous substitutions in the extracellular do- cellular domain of the LH/CG receptor for the corresponding main of the human TSH receptor with segments of the rat region ofthe TSH receptor resulted in high-affinity human CG LH/CG receptor. The chimeric TSH-LH/CG receptors con- binding with complete loss of TSH binding. Surprisingly, structed reveal that multiple regions in the middle region and however, there was at least one chimera with a substitution at carboxyl-terminal half (residues 171-418) ofthe extracellular each of the five domains that still retained high-affinity TSH domain are involved in signal transduction. In addition, the binding. Substitution of residues 1-170 of the TSH receptor TSH-binding region is likely to span the entire extracellular with the corresponding region of the LH/CG receptor was domain, with multiple discontinuous contact sites. There associated with the retention of high-affinity TSH binding but appears to be considerable tolerance for alteration in the ligand specificity was lost in that TSH and human CG could hormone-binding region. interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the MATERIALS AND METHODS extracellular domain ofthe TSH receptor are involved in signal Construction and Functional Expression of Chimeric TSH- transduction and that the TSH-binding region is likely to span LH/CG Receptor cDNAs. Chimeras were constructed using the entire extracellular domain, with multiple discontinuous five restriction endonuclease sites in the extracellular domain contact sites. of the full-length human TSH receptor cDNA in Bluescript (1) (SnaBI at amino acid 82, Mlu I at amino acid 170, Afi II The human thyrotropin (TSH) receptor (1-3) as well as other at amino acid 260, EcoRV at amino acid 360, and Spe I at pituitary [luteinizing hormone (LH) and follicle-stimulating amino acid 418) and the Sal I site in the multiple cloning site hormone (FSH)] and placental [chorionic gonadotropin (CG)] of the vector. Two of the sites in the TSH receptor cDNA glycoprotein hormone receptors (4-6) belong to a subgroup (SnaBI and Afl II) were preexisting restriction sites. The of the guanine nucleotide (G) regulatory protein-coupled other three sites (Mlu I, EcoRV, and Spe I) were chosen by receptor family with very large extracellular domains. With their uniqueness to the plasmid containing the cDNA and 14 incomplete leucine-rich repeated segments this subgroup's were created by using oligonucleotide-directed mutagenesis receptors are also members of the leucine-rich glycoprotein (Bio-Rad Muta-Gene Phagemid in vitro mutagenesis kit) (18). family (7-11). The unusually large size of the extracellular These new sites created two conservative amino acid sub- domain (418 amino acids) of the TSH receptor (764 amino stitutions (Glu -+ Asp at residue 362 and Ile -- Leu at residue acids), the very large size (28 kDa) ofits specific ligand, TSH, 419). The TSH receptor cDNA with the two conserved amino as well as cross-linking studies (12) make it likely that TSH acid substitutions was excised with EcoRI and subcloned into binds to the extracellular domain of this receptor. Indeed, the expression vector pSV2-NEO-ECE (1). human CG (hCG) binds with high affinity to the extracellular Rat LH/CG receptor cDNA was synthesized by the poly- domain of the LH/CG receptor (6). There have been no merase chain reaction (PCR) (19) using as template -10 ng of studies to localize in these large extracellular domains the Abbreviations: TSH, thyrotropin; LH, luteinizing hormone; CG, The publication costs of this article were defrayed in part by page charge chorionic gonadotropin; hCG, human CG; FSH, follicle-stimulating payment. This article must therefore be hereby marked "advertisement" hormone; CHO, Chinese hamster ovary; PCR, polymerase chain in accordance with 18 U.S.C. §1734 solely to indicate this fact. reaction.

902 Downloaded by guest on September 24, 2021 Medical Sciences: Nagayarna et al. Proc. NatL. Acad. Sci. USA 88 (1991) 903 phage DNA extracted from a rat ovarian library (Clontech) was determined in the presence of 1 ,uM TSH and this value and two oligonucleotides containing the appropriate restric- was subtracted from total binding to yield specific TSH tion sites at their5' ends. These restriction sites were then used binding. hCG binding was determined as for TSH with the for substitution ofthe LH/CG receptor cDNA fragments into exceptions that we used the conditions of Buettner and TSH receptor cDNA from which the corresponding region had Ascoli (23). As ligand we used highly purified hCG (11,000 been deleted using the same restriction enzymes. The nucle- units/mg of protein) radiolabeled with 1251 by the stoichio- otide sequences of the PCR-generated fragments of the rat metric chloramine-T method (24) to a specific activity of 50 LH/CG receptor as well as the ligation sites and adjacent ,uCi/,g of protein. Measurements of the intracellular cAMP nucleotide sequences of the TSH receptor in the chimeric response to hormone stimulation (1 hr at 37°C) were as constructs were determined (20) in full and compared to the described (25). previously published sequence data (1, 4). Only chimeric cDNAs without any amino acid substitutions were chosen. RESULTS The chimeric TSH-LH/CG cDNA was transfected into To conserve the three-dimensional structure of the TSH Chinese hamster ovary (CHO) cells by the calcium-phos- receptor we constructed 11 chimeric human TSH-rat LH/CG phate method (21). Surviving colonies were selected by G418 receptor cDNAs (Fig. 1), which were then stably expressed (400 1g/ml) and pooled for study of their ability to bind to in CHO cells. For chimeric receptor construction we divided TSH and hCG and to respond to TSH and hCG with respect the extracellular domain ofthe human TSH receptor into five to an increase in intracellular cAMP levels. regions by restriction endonuclease sites, three ofwhich sites TSH and hCG Binding and Intracellular cAMP Measure- were introduced by site-directed mutagenesis. One or more ments. TSH-binding studies were performed as described (22) ofthese regions were replaced with the homologous region of with the exception that highly purified bovine TSH (30 the rat LH/CG receptor. Pools ofstably transfected clones of units/mg of protein) was iodinated with 125I to =80 ,uCi/,ug cells were tested for their ability to bind to TSH and hCG and of protein using the Bolton-Hunter reagent (4400 Ci/mmol; to respond to TSH and hCG stimulation in terms of an 1 Ci = 37 GBq; New England Nuclear). In addition, we tested increase in intracellular cAMP levels. competition for 1251-labeled TSH (125I-TSH) binding with The human TSH receptor with two conserved amino acid hCG as well as TSH (Sigma). Nonspecific 125I-TSH binding substitutions (introduced with the three new restriction sites EXTRACELLULAR DOMAIN

SnaB Mlu I Afi 11 EcoR V Speel RECEPTOR LRCG TSH A I B I c I D I E RECEPTOR 1 82 170 260 360 4118 SUBSTITUTION

TSH-LHR-1 1 -83

TSH-LHR-2 I I 84-171 TSH-LHR-3 I _- I 172-260

TSH-LHR-4 261-316

TSH-LHR-5 I_ 317-367 TSH-LHR-6 I 261-367 TSH-LHR-7 I 172-367

TSH-LHR-8 1-171

TSH-LHR-9 1 -260 1-171, TSH-LHR-10 317-367

TSH-LHR-1 1 1-367

FIG. 1. Schematic representation of chimeric TSH-LH/CG receptor extracellular domains. The 418-amino acid extracellular region of the human TSH receptor (764 amino acids) was divided into five arbitrary domains (A-E) on the basis of the indicated restriction sites. In the chimeras, the open areas denote the region(s) ofthe extracellular domain of the human TSH receptor that remained after substitution. The solid areas depict the homologous regions ofthe rat LH/CG receptor that were used to substitute for the deleted TSH receptor domains. The LH/CG receptor fragment inserted into TSH receptor domain D is 50 amino acids smaller than its TSH receptor counterpart, corresponding to the nature of the normal LH/CG receptor in this region (4), as shown by the horizontal line. Chimeric receptors are designated as TSH-LHR-1 through TSH-LHR-11. The numbers assigned to the amino acids are those published for the human TSH receptor (1) and for the rat LH/CG receptor (4). Signal sequences have not been deleted. The identities of the LH/CG receptor fragments inserted into the indicated restriction sites in the TSH receptor are indicated. Downloaded by guest on September 24, 2021 904 Medical Sciences: Nagayama et al. Proc. NatL Acad. Sci. USA 88 (1991)

for chimera construction) was identical to the wild-type in the functional activity of these two chimeras. Thus, the receptor (22) in terms of its affinity for TSH and its ability to cellular cAMP response to TSH stimulation with TSH-LHR-8 mediate an increase in intracellular cAMP levels (data not was similar to that of the wild-type TSH receptor (Table 1). shown). Chimera TSH-LHR-11, in which the entire extra- Consistent with the binding data, hCG also increased intra- cellular domain of the LH/CG receptor replaced the corre- cellular cAMP levels in TSH-LHR-8. In contrast, chimera sponding region of the TSH receptor, showed high-affinity TSH-LHR-9 was functionally unresponsive to TSH and hCG hCG binding comparable to that reported for the wild-type stimulation. This complete dissociation between ligand bind- LH/CG receptor (4, 23) as well as a cAMP response to hCG ing and receptor function in TSH-LHR-8 and TSH-LHR-9 stimulation (Fig. 1 and Table 1). This chimera did not implicates domain C (residues 171-260) (Fig. 1) in the trans- demonstrate high-affinity TSH binding. These data indicate duction of a signal by the TSH receptor. that, as anticipated, the extracellular domains of the glyco- Focusing on the carboxyl terminus of the TSH receptor protein hormone receptors are critical for their respective extracellular domain, with chimera TSH-LHR-6 (residues ligand binding. Of the chimeric receptors in which only a 261-418), a more extensive substitution than TSH-LHR-5 single TSH receptor region was replaced (chimeras TSH- (see above), TSH still bound with high affinity similar to the LHR-1 through TSH-LHR-5), chimeras TSH-LHR-1 (sub- wild-type receptor (Table 1). However, in contrast to their stitution of TSH receptor residues 1-82) and TSH-LHR-3 normal TSH binding, the signal transduction ofthese mutant (residues 171-260) lost their high-affinity TSH-binding site. receptors was impaired but not abolished. Further substitu- In contrast, chimeras TSH-LHR-2 (residues 83-170), TSH- tion of the carboxyl region of the TSH receptor extracellular LHR-4 (residues 261-360), and TSH-LHR-5 (residues 361- domain (TSH-LHR-7; residues 171-418) abolished high- 418) bound TSH with high affinity. As with the wild-type affinity TSH binding. TSH receptor (22), hCG did not bind to chimeras TSH- Substitutions ofamino-terminal and carboxyl-terminal seg- LHR-2, TSH-LHR-4, and TSH-LHR-5, indicating that these ments ofthe TSH receptor in chimera TSH-LHR-10 (residues chimeras retained their specificity for TSH binding. Func- 1-170 and 361-418) did not alter high-affinity TSH binding tionally, as determined by the intracellular cAMP response to (Table 1) in accordance with their individual substitutions TSH stimulation (Table 1) chimeras TSH-LHR-1 and TSH- (TSH-LHR-8 and TSH-LHR-5). Chimera TSH-LHR-10 LHR-3 were totally inactive, as expected in view of their bound hCG with a relatively low affinity similar to TSH- inability to bind the hormone with high affinity. In contrast, LHR-8. Surprisingly, however, the sensitivity of the cAMP chimeras TSH-LHR-4 and TSH-LHR-5, with high affinities response to TSH stimulation of TSH-LHR-10 was increased for TSH binding, had blunted cAMP responses to TSH 5-fold relative to the wild-type TSH receptor and contrasted stimulation. TSH-LHR-2 had normal high-affinity TSH bind- with the reduced signal transduction with the isolated E ing and a normal cAMP response to TSH stimulation. None domain substitution in chimera TSH-LHR-5. of these chimeras responded to hCG stimulation. In the chimeras that bound TSH and hCG (chimeras TSH- The foregoing data suggested that the carboxyl-terminal LHR-8, TSH-LHR-9, and TSH-LHR-10), to evaluate whether region ofthe TSH receptor extracellular domain played a role hCG was interacting with the chimeric receptors in the same in signal transduction and that amino acid residues in two region as TSH we also tested competition by hCG for 125I-TSH discontinuous regions (1-82 and 171-260) in the TSH receptor binding. In all three chimeras, hCG competed for 125I-TSH extracellular region were important for TSH binding. Surpris- binding but only with low affinity (data not shown). ingly, however, more extensive substitutions of these regions in chimeras TSH-LHR-8 (residues 1-170) and TSH-LHR-9 DISCUSSION (residues 1-260, representing 62% ofthe extracellular domain) It is well-established that the intracellular, cytoplasmic do- were associated with high-affinity TSH binding (Table 1). mains of other members of the G protein-coupled receptor Furthermore, these chimeras also lost their complete speci- family play an important role in signal transduction (26, 27). ficity for TSH and also interacted with hCG, though with a The majority of these receptors (which we term subgroup A) lower affinity than for chimera TSH-LHR-11 containing the interact with very small ligands and lack significant extra- entire LH/CG extracellular domain. Remarkably, despite cellular domains. The present data with the TSH receptor, a their similar, high-affinity TSH binding, there was a dichotomy member of this receptor family with substantial extracellular Table 1. Hormone binding and function in CHO cells stably expressing the TSH-LH/CG chimeras depicted in Fig. 1 Hormone binding cAMP response TSH hCG TSH hCG Chimeric Kd (M) Kd (M) EC"0 (M) EC50 (M) receptor Kd, nM Kd (Wt) Kd, nM Kd (Wt)* EC50, nM EC50 (Wt) EC50, nM EC50 (Wt)t Wt 0.3 1 ND ND 4.0 1 ND ND TSH-LHR-1 ND ND ND ND ND ND ND ND TSH-LHR-2 0.6 2 ND ND 6.5 1.6 ND ND TSH-LHR-3 ND ND ND ND ND ND ND ND TSH-LHR-4 0.2 0.7 ND ND 23 5.8 ND ND TSH-LHR-5 0.2 0.7 ND ND 20 5.0 ND ND TSH-LHR-6 0.2 0.7 ND ND 28 7.0 ND ND TSH-LHR-7 ND ND ND ND ND ND ND ND TSH-LHR-8 0.2 0.7 3.4 113 4.8 1.2 20 50 TSH-LHR-9 0.05 0.2 24 800 ND ND ND ND TSH-LHR-10 0.1 0.3 8 267 0.9 0.2 24 60 TSH-LHR-11 ND ND 0.03 1 ND ND 0.4 1 M, mutant; Wt, wild type; ND, not detectable. Each value represents the mean of data obtained with pools of clones from two separate transfections, each transfection measured in duplicate. *Relative to the Kd for hCG binding to TSH-LHR-11. tRelative to the EC50 for hCG stimulation of TSH-LHR-11. Downloaded by guest on September 24, 2021 Medical Sciences: Nagayarna et al. Proc. Natl. Acad. Sci. USA 88 (1991) 905 domains for large ligands (subgroup B), suggest that the mation a practical prerequisite for future detailed mutagen- middle region and carboxyl half of the extracellular domain esis studies to define more precisely the amino acid residues of the TSH receptor (domains C, D, and E) (Fig. 1) play an involved in hormone binding and signal transduction. important role in signal transduction. Domain C appears to be particularly dominant. This mechanism would differ from the We thank Drs. Fred Cohen and Scott Pressley for helpful discus- A subgroup of receptors (such as the adrenergic receptors), sions during the course ofthis work and also Mr. Gil dela Calzada for in which the ligand interacts directly with transmembrane his expert administrative assistance. This research was supported by regions. The data with chimera TSH-LHR-10 also raise the National Institutes ofHealth Grants DK 19289 and DK 36182 and the possibility of an interaction between discontinuous amino- Research Service of the Veterans Administration. and carboxyl-terminal regions of the receptor extracellular domain (domains A, B, and E) in the process of signal 1. Nagayama, Y., Kaufman, K. D., Seto, P. & Rapoport, B. It and (1989) Biochem. Biophys. Res. Commun. 165, 1184-1190. transduction. is also interesting that the amino car- 2. Libert, F., Lefort, A., Gerard, C., Parmentier, M., Perret, J., boxyl regions ofthe extracellular domain ofthe TSH receptor Ludgate, M., Dumont, J. E. & Vassart, G. (1989) Biochem. are rich in cysteine residues that are conserved among Biophys. Res. Commun. 165, 1250-1255. members of receptor subgroup B (1, 4-6, 28), and there is 3. Misrahi, M., Loosfelt, H., Atger, M., Sar, S., Guiochon- evidence for the existence of disulfide-linked subunits in the Mantel, A. & Milgrom, E. (1990) Biochem. Biophys. Res. TSH receptor (12). It is conceivable, therefore, that disulfide Commun. 166, 394-403. bonds may maintain contact between extracellular domains 4. McFarland, K. C., Sprengel, R., Phillips, H. S., Kohler, M., A and E in the process of signal transduction. Rosemblit, N., Nikolics, K., Segaloff, D. L. & Seeburg, P. H. With respect to hormone binding, substitution ofthe entire (1989) Science 245, 494-499. LH/CG receptor extracellular domain for the corresponding 5. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Vu Hai-Luu region ofthe TSH receptor (chimera TSH-LHR-11) indicates Thi, M. T., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, that the extracellular domain of the glycoprotein hormone B., Gamier, J. & Milgrom, E. (1989) Science 245, 525-528. receptors is and for Nev- 6. Sprengel, R., Braun, T., Nikolics, K., Segaloff, D. L. & sufficient, critical, ligand binding. Seeburg, P. H. (1990) Mol. Endocrinol. 4, 525-530. ertheless, it is remarkable that no single domain in the 7. Takahashi, N., Takahashi, Y. & Putnam, F. W. (1985) Proc. extracellular region ofthe TSH receptor can be implicated as Nati. Acad. Sci. USA 82, 1906-1910. being a dominant site for high-affinity TSH binding-that is, 8. Ferrero, E., Hsieh, C., Francke, U. & Goyert, S. M. (1990) J. there is at least one chimeric receptor with a substitution for Immunol. 145, 331-336. every segment of the TSH receptor extracellular domain 9. Krusius, T. & Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. USA (A-E) that binds to TSH with high affinity. It seems likely 83, 7683-7687. that the absence of high-affinity TSH binding with 3 of the 11 10. Hashimoto, C., Hudson, K. L. & Anderson, K. V. (1988) Cell chimeras (TSH-LHR-1, TSH-LHR-3, and TSH-LHR-7) may 52, 269-279. reflect abnormal folding or instability ofthe chimeric receptor 11. Lopez, J. A., Chung, D. W., Fujikawa, K., Hagen, F. S., proteins because substitution of the same regions in other Papayannopoulou, T. & Roth, G. J. (1987) Proc. Natl. Acad. chimeras did not affect TSH binding. We interpret these Sci. USA 84, 5615-5619. to that the TSH on 12. Buckland, P. R., Rickards, C. R., Howells, R. D., Jones, findings suggest binding site the receptor E. D. & Rees Smith, B. (1982) FEBS Lett. 145, 245-249. is likely to be discontinuous, with multiple contact points 13. Cunningham, B. C., Henner, D. J. & Wells, J. A. (1990) Sci- between the two molecules. Unlike the growth hormone- ence 247, 1461-1465. hormone family (13, 14), homologous substitutions 14. Cunningham, B. C., Jhurani, P., Ng, P. & Wells, J. A. (1989) in a limited number of sites in the TSH receptor are tolerated Science 243, 1330-1336. without significant change in high-affinity ligand binding. 15. Laver, W. G., Air, G. M., Webster, R. G. & Smith-Gill, S. J. The present data provide information not only about the (1990) Cell 61, 553-556. TSH receptor but also about the LH/CG receptor. Unlike 16. Cunningham, B. C. & Wells, J. A. (1989) Science 244, 1081- with the TSH receptor, none ofthe chimeras studied retained 1085. hCG even some 17. Finke, R., Seto, P. & Rapoport, B. (1990) J. Clin. Endocrinol. high-affinity binding, though did interact Metab. 71, 53-59. with this hormone with relatively low affinity. Therefore, the 18. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. LH/CG receptor appears to be less tolerant of homologous 19. Saiki, R. K., Gelfand, D. N., Stoffel, S., Scharf, S. J., Higuchi, substitution than the TSH receptor. R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science The chimeric receptor approach used in this study is a 239, 487-491. powerful means to define domains important for the unique 20. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. functions of members of a homologous protein family. How- Acad. Sci. USA 74, 5463-5467. ever, it does not identify regions that may be involved in a 21. Chen, C. & Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752. function common to all members of the protein family. For 22. Chazenbalk, G. D., Nagayama, Y., Kaufman, K. D. & Rapo- the have a common a port, B. (1990) Endocrinology 127, 1240-1244. example, glycoprotein 23. Buettner, K. & Ascoli, M. (1984) J. Biol. Chem. 259, 15078- subunit that binds to the extracellular domain ofthe receptor. 15084. This common region would not be identified by the homol- 24. Goldfine, I. D., Amir, S. M., Petersen, A. W. & Ingbar, S. H. ogous substitution approach used. Indeed, mutagenesis stud- (1974) Endocrinology 95, 1228-1233. ies of an 8-amino acid region (amino acid residues 38-45) in 25. Hirayu, H., Magnusson, R. P. & Rapoport, B. (1985) Mol. Cell. the human TSH receptor suggest that this region is critical for Endocrinol. 42, 21-27. TSH binding (29). Because homologous substitution of this 26. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., region did not abolish high-affinity TSH binding (present Caron, M. G. & Lefkowitz, R. J. (1988) Science 240, 1310- study), it is likely to be a site of interaction with the common 1316. a of the 27. Strader, C. D., Sigal, I. S. & Dixon, R. A. F. (1989) FASEB J. subunit glycoprotein hormones. 3, 1825-1832. In conclusion, the present data provide delineation of 28. Parmentier, M., Libert, F., Maenhaut, C., Lefort, A., Gerard, functional domains in the extracellular component of a mem- C., Perret, J., Van Sande, J., Dumont, J. E. & Vassart, G. ber of the newly defined group of G protein-coupled recep- (1989) Science 246, 1620-1622. tors with large extracellular domains (subgroup B). The large 29. Wadsworth, H. L., Chazenbalk, G. D., Nagayama, Y., Russo, extracellular domain of the TSH receptor makes this infor- D. & Rapoport, B. (1990) Science 249, 1423-1425. Downloaded by guest on September 24, 2021