Single-Chain, Triple-Domain Gonadotropin Analogs with Disulfide Bond Mutations in the α-Subunit Elicit Dual Follitropin and Lutropin Activities in Vivo

Abstract

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

FSHβ knockout mouse model: a decade ago and into the future

“…The gonadotrope-targeted hFSH β transgene rescued Fshb null male and female mice, and ectopically produced hFSH rescued null males but only partially rescued female Fshb null mice [25]. These mice have been extensively used later on in collaborative studies with Dr. Irving Boime, to test the bioactivities of various gonadotropin analogs [26, 27]. …”

The Gonadotropin Hormones and Their Receptors∗

“…76,77 Subsequently, similar fusion proteins of LH and FSH were also found to be bioactive. [82][83][84] The single chain hormones also displayed increased stability and heat resistance in vitro compared to their heterodimeric counterparts. 80,81 From these studies it was concluded that the N-α-hCGβ-C configuration was also bioactive and, quite surprisingly, that, each disulfide of the subunits could be eliminated without a loss of activity.…”

“…90 A second single chain hCG antagonist was designed by mutating three of the four N-linked glycosylation sites that are associated with LHR activation (Asn 13 and 30 in the β subunit and Asn 52 in the α subunit). A most provocative finding was based on a bifunctional, triple domain fusion protein of the form: N-FSHβ-hCGβ-α-C. 84 These investigators showed that disruption of heterodimer formation by mutation of either Cys10-Cys60 or Cys32-Cys84 did not eliminate bioactivity, and thus concluded that αβ contacts are not required for receptor binding and activation. This analog behaved as a competitive antagonist and suppressed ovarian hyperstimulation syndrome in rats.…”

A Carboxyl-terminal Sequence in the Lutropin β Subunit Contributes to the Sorting of Lutropin to the Regulated Pathway

“…Those analyses used polyclonal antisera to FSH/FSH␤. Here we used a high affinity FSH dimerspecific monoclonal antibody (23,24) to identify the proteins because it does not cross-react with the uncombined FSH␤ subunit, thus reducing the background. As shown previously, the ␣ and FSH␤ subunits migrate at similar rates on SDS gels, and their resolution is not clear (21).…”