Thyrotropin, Follitropin, and Chorionic Gonadotropin Expressed as a Single Multifunctional Unit Reveal Remarkable Permissiveness in Receptor-Ligand Interactions

Campayo, Javier García; Kumar, T. Rajendra; Boime, Irving

doi:10.1210/en.2002-220336

Cited by 26 publications

(23 citation statements)

References 46 publications

(42 reference statements)

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“…This indicates a tremendous degree of flexibility in the hormone receptor interactions and shows that the receptor has the ability to accommodate analogs with altered conformation as seen in the cases of phCGαP38Aβ and phCGαT54Aβ. A similar degree of plasticity of glycoprotein-hormone receptors has been reported in several studies involving analogs with compromised subunit interaction [38] and multi-subunit derivatives [39][40][41][42]. All the analogs synthesized in these studies were shown to be biologically active, demonstrating that a heterodimer-like conformation is not an absolute necessity for receptor interaction and that as long as the key hormone-receptor contacts are maintained, the analogs can be accommodated by the receptor.…”

Section: Discussionsupporting

confidence: 68%

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

Setlur

Dighe

2006

Glycoconj J

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The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the alpha and beta subunits of human Chorionic Gonadotropin (hCG) to produce a single chain hormone (phCGalphabeta) was used to investigate roles of critical residues of the alpha subunit in hormone receptor interaction and biological activity. The alpha subunit was mutated using PCR-based site-directed mutagenesis, fused to the wild type beta subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological probes, receptor assays, and in vitro bioassays. The mutation hCGalpha P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower affinity and elicit biological response. Mutation of hCGalpha T54A disrupting the glycosylation at Asparagine 52, believed to be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the alpha subunit led to the generation of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone, and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and elicit response. These data suggest the plasticity of the hormone-receptor interactions.

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Section: Discussionsupporting

confidence: 68%

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

Setlur

Dighe

2006

Glycoconj J

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“…For example, a tetradomain glycoprotein hormone analog consisting of TSHb, FSHb, hCGb and a-subunits tandemly linked was expressed in vitro. This analog elicited multiple hormone activities in vivo and pharmacologically rescued FSHb knockout mice (Garcia-Campayo et al 2002, GarciaCampayo et al 2005. It may prove useful for studying thyroid regulation of the fertility status in vivo during normal and abnormal reproductive cycles.…”

Section: Discussionmentioning

confidence: 99%

What have we learned about gonadotropin function from gonadotropin subunit and receptor knockout mice?

Kumar¹

2005

Reproduction

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A number of biochemical and physiological studies elucidated the roles of pituitary and placental glycoprotein hormones. Advances in the past two decades in manipulating the mouse genome by random or site-specific mutagenesis have heralded a new dimension to our understanding of the biology of gonadotropins. It is now possible to model many human reproductive disorders involving gonadotropins/gonadotropin-signaling in the mouse. Mutant mice selectively lacking either FSH or LH or their cognate receptors have been generated. The gonadotropin ligand and the corresponding receptor knockout mice mostly phenocopy each other. Analyses with these genetic models confirmed earlier physiological studies; in addition they also revealed novel roles for gonadotropins previously unrecognized. While FSH action seems dispensable for male but not female fertility, absence of LH causes infertility in both the sexes. While Sertoli cell number and germ cell carrying capacity of the Sertoli cells in compromised in FSH mutants, both somatic and germ cell lineages are affected in the LH mutants resulting in complete male infertility. FSH mutant females demonstrate a pre-antral stage block in folliculogenesis and FSH alone is not sufficient to promote full folliculogenesis in the absence of LH. Pre-ovulatory stage follicles do not form and most of the follicles undergo apoptosis in the absence of LH. Many extra-gonadal phenotypes have been described for the receptor knockout mice and whether these bear any resemblances to those in patients with similar inactivating mutations in the receptors for FSH and LH remains an open question. Thus the in vivo models will continue to have a significant impact in understanding gonadotropin physiology and pathophysiology and serve as novel genetic tools to study signaling mechanisms in the gonads.

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“…However, this approach precluded detailed structure-function analyses due to mutagenesis-induced defects in subunit assembly [3,7]. To bypass the subunit-assembly step, the ␣ and ␤ subunits were genetically fused, resulting in novel single-chain glycoprotein analogs [8][9][10][11]. Earlier studies demonstrated successful generation of an analogue in which one ␣ chain was fused in tandem with one or more ␤ subunits [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

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

Campayo

Boime

Ma³

et al. 2005

Biology of Reproduction

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We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific beta subunit genes to the common alpha 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) beta, follicle-stimulating hormone (FSH) beta, and chorionic gonadotropin (CG) beta subunits to the alpha 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 FSHbeta 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.

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Thyrotropin, Follitropin, and Chorionic Gonadotropin Expressed as a Single Multifunctional Unit Reveal Remarkable Permissiveness in Receptor-Ligand Interactions

Cited by 26 publications

References 46 publications

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

Single chain human chorionic gonadotropin, hCGαβ: Effects of mutations in the α subunit on structure and bioactivity

What have we learned about gonadotropin function from gonadotropin subunit and receptor knockout mice?

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

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