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A Single-Chain Tetradomain Glycoprotein Hormone Analog Elicits Multiple Hormone Activities In Vivo¹

Department of Molecular Biology and Pharmacology,³ Washington University School of Medicine, St. Louis, Missouri 63110 Departments of Pathology⁴ Molecular and Cellular Biology,⁵ Baylor College of Medicine, Houston, Texas 77030

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific ß subunit genes to the common subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) ß, follicle-stimulating hormone (FSH) ß, and chorionic gonadotropin (CG) ß subunits to the subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHß knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

¹ Supported in part by the Department of Pathology Startup Funds, The Moran Foundation (to T.R.K.), and a research grant from Johnson and Johnson (to I.B.). A part of the work reported in this manuscript has been presented at the 36th Annual Meeting of the Society for the Study of Reproduction, Cincinnati, OH (2003).

² Correspondence and current address: T. Rajendra Kumar, Department Molecular and Integrative Physiology, University of Kansas Medical Center, 3011 WHE, Mailstop 3043, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 0455; tkumar{at}kumc.edu

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				A. Jablonka-Shariff, T. R. Kumar, J. Eklund, A. Comstock, and I. Boime Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the {alpha}-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo Mol. Endocrinol., June 1, 2006; 20(6): 1437 - 1446. [Abstract] [Full Text] [PDF]

				T R. Kumar What have we learned about gonadotropin function from gonadotropin subunit and receptor knockout mice? Reproduction, September 1, 2005; 130(3): 293 - 302. [Abstract] [Full Text] [PDF]