Receptor-selective determinants in catfish gonadotropin seat-belt loops

doi:10.1016/j.mce.2004.06.011

Molecular and Cellular Endocrinology

Volume 224, Issues 1–2, 30 September 2004, Pages 55-63

https://doi.org/10.1016/j.mce.2004.06.011 Get rights and content

Abstract

Mammalian gonadotropins are highly selective. Charge differences between the Cys^10–11 sequence of FSHβ and LHβ/CGβ seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHβ-specific sequence between Cys^11–12 of the seat-belt loop. Intriguingly, African catfish LHβ (cfLHβ) lacks a positively charged Cys^10–11 region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys^10–11 region of cfLHβ contains cfLH-R-selective determinants, whereas the Cys^11–12 region of cfLHβ confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure–function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

Introduction

In mammals, gonadal function is regulated by two distinct, but complementary acting, pituitary-derived gonadotropins (follicle-stimulating hormone, FSH, and luteinizing hormone, LH) via the specific activation of their respective receptors (FSH-R and LH-R). LH and FSH, together with thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG), form the family of glycoprotein hormones, which are glycosylated heterodimeric molecules, each composed of a non-covalent association of a common α-subunit with a hormone specific β-subunit. Apart from LH and CG that both are able to activate the LH-R in primates and equids, the normal interaction between the glycoprotein hormones and their respective receptors are highly specific (<0.1% cross-reactivity; Campbell et al., 1997, Vischer et al., 2003a). In contrast to mammalian gonadotropins, their counterparts in fish display reduced receptor selectivity. For example, purified, pituitary-derived catfish LH (cfLH) as well as recombinant cfLH (rcfLH) can activate both the catfish LH receptor (cfLH-R; Vischer and Bogerd, 2003a) and catfish FSH receptor (cfFSH-R; Bogerd et al., 2001) with comparable potencies. However, cfLH is not able to activate the catfish TSH-R (Vischer and Bogerd, 2003b). Moreover, recombinant catfish FSH (rcfFSH) predominantly activates the cfFSH-R (Vischer et al., 2003b).

Crystallographic analyses of deglycosylated hCG and hFSH (Lapthorn et al., 1994, Fox et al., 2001) suggested a similar overall folding of the common α-subunit, but also of the two gonadotropin β-subunits, each consisting of a cystine knot architecture that divides each subunit into three elongated antiparallel loops (i.e. α1, α2, α3; β1, β2, β3). Although the primary β-subunit sequences have diverged sufficiently during evolution to confer specificity to each of these heterodimeric glycoprotein hormones (Li and Ford, 1998), their β-subunits are structurally very similar due to the positional conservation of 12 conserved cysteine residues. Six cysteines form the cystine knot motif, while the remaining six residues form three additional loop-stabilizing disulfide bridges.

Based on sequence differences between distinct β-subunits, chimeric analogs have been generated in order to identify β-subunit regions that confer receptor-specificity to these hormones. These studies revealed that the region between the 10th and 12th conserved cysteine residues (Cys^10–12; i.e. the seat-belt region) of each glycoprotein hormone β-subunit is critically involved in determining specificity for its respective receptor. In particular, the net charge differences in the determinant loop (i.e. the region between Cys¹⁰ and Cys¹¹) of the seat-belt region between mammalian LH/CG β-subunits on the one hand, and FSH/TSH β-subunits on the other hand are thought to have partially separated LH-R- from FSH-R/TSH-R-activating properties (Han et al., 1996, Campbell et al., 1997). It is thought that additional sequence divergence in the carboxy-terminal seat-belt segment (i.e. between Cys¹¹ and Cys¹²) as well as outside the seat-belt region has further separated specific lutropic, follitropic and thyrotropic activities of these hormones to their respective natural receptors (Grossmann et al., 1997).

In contrast to the situation in mammalian gonadotropins, inspection of the determinant loop of fish gonadotropins revealed the absence of striking net charge differences between their LH and FSH β-subunits. To elucidate if and how the seat-belt region of cfLHβ contributes to the observed promiscuous receptor activation (Bogerd et al., 2001, Vischer and Bogerd, 2003a), we studied the effects of several substitutions of the intercysteine segments of the cfLHβ seat-belt region in order to identify sequence information necessary to specifically stimulate the cfFSH-R and the cfLH-R.

Section snippets

Construction and expression of cassette-substituted gonadotropin cDNAs

Chimeric seat-belt cfLH β-subunit (cfLHβ) constructs were generated by cassette-substitution of individual intercysteine segments of the seat-belt region or the entire seat-belt region between the 10th and 12th conserved cysteine residues (i.e. Cys⁹⁰ and Cys¹⁰⁷, respectively) with the corresponding hCG-regions or cfFSH-regions, or with the corresponding number of Ala residues (Fig. 1). In addition, reciprocal chimeras were made by introducing the cfLH seat-belt region sequences into the cfFSH

Catfish gonadotropin expression in Dictyostelium

Recombinant mutant gonadotropins, expressed in Dictyostelium discoideum, were first functionally analyzed (see below), and a posteriori quantified by radioimmunoassay in order to limit the storage time between the concentration procedure and the functional assays. Recombinant gonadotropin levels were measured by two different radioimmunoassays using either a polyclonal antibody against intact cfLH, or a polyclonal antibody against the cfGP α-subunit, as described previously (Vischer et al.,

Discussion

Glycoprotein hormones have coevolved together with their cognate receptors through gene duplications of ancestral β-subunit and receptor genes, respectively, followed by sequence divergence to define hormone-receptor pair selectivity (Moyle et al., 1994, Li and Ford, 1998, Moyle et al., 1998). However, functional duality of gonadotropins has not always evolved simultaneously with their structural divergence. To investigate if and how the seat-belt loop of cfLHβ allows the interaction of cfLH

References (33)

F. Chen et al.
Role of the invariant aspartic acid 99 of human choriogonadotropin β in receptor binding and biological activity
J. Biol. Chem.
(1991)
W. Chen et al.
A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways
Anal. Biochem.
(1995)
J.A. Dias et al.
Receptor binding and functional properties of chimeric human follitropin prepared by an exchange between a small hydrophylic intercysteine loop of human follitropin and human lutropin
J. Biol. Chem.
(1994)
M. Grossmann et al.
Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) β-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH
J. Biol. Chem.
(1997)
Y. Han et al.
hCGβ residues 94–96 alter LH activity without appearing to make key receptor contacts
Mol. Cell. Endocrinol.
(1996)
J. Huang et al.
Mutagenesis of the determinant loop region of human choriogonadotropin
Mol. Cell. Endocrinol.
(1993)
Y. Koide et al.
Maturational gonadotropin from the African catfish (Clarias gariepinus): purification, characterization, localization and biological activity
Gen. Comp. Endocrinol.
(1992)
W.R. Moyle et al.
Functional homodimeric glycoprotein hormones: implications for hormone action and evolution
Chem. Biol.
(1998)
B. Quérat et al.
Duality of gonadotropins in Gnathostomes
Gen. Comp. Endocrinol.
(2001)
K. Takada et al.
Specific gonadotropin binding sites in the bullfrog testis
Gen. Comp. Endocrinol.
(1986)

H.F. Vischer et al.

Ligand selectivity of gonadotropin receptors. Role of the β-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor

J. Biol. Chem.

(2003)

N. Wakabayashi et al.

The cloning and transient expression of a chicken gene encoding a follicle-stimulating hormone receptor

Gene

(1997)

S.A. Ali et al.

PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations

Biotechniques

(1995)

J. Bogerd et al.

Discrepancy between molecular structure and ligand selectivity of a testicular FSH receptor of the African catfish (Clarias gariepinus)

Biol. Reprod.

(2001)

R.K. Campbell et al.

Conversion of human choriogonadotropin into follitropin by protein engineering

Proc. Natl. Acad. Sci. U.S.A.

(1991)

R.K. Campbell et al.

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein designs

Nat. Biotech.

(1997)

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Further separation of specific FSH, TSH and LH activities has been shown by additional sequence divergence in the carboxy-terminal seatbelt segment as well as outside the seatbelt region (Grossmann et al., 1997). Similar domain exchange studies have been performed only for African catfish gonadotropins (Vischer et al., 2004), and revealed that the determinant loop of African catfish LHβ (cfLHβ) is essential for its LH activity (directing the hormone’s activity only to the LHR), but seems to be dispensable for its FSHR-stimulating activity. Whereas the negatively charged surface of the determinant loop of human FSHβ acts as an inhibitory determinant by blocking unintended interactions with the LHR (Dias et al., 1994; Lindau-shepard et al., 1994), the determinant loop of African catfish FSHβ (cfFSHβ), although essential for its FSHR-stimulating activity, does not seem to play such an inhibiting role to block interaction with the LHR (Vischer et al., 2004).
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¹: Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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Receptor-selective determinants in catfish gonadotropin seat-belt loops

Abstract

Introduction

Section snippets

Construction and expression of cassette-substituted gonadotropin cDNAs

Catfish gonadotropin expression in Dictyostelium

Discussion

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Mol. Cell. Endocrinol.

Gen. Comp. Endocrinol.

Chem. Biol.

Gen. Comp. Endocrinol.

Gen. Comp. Endocrinol.

J. Biol. Chem.

Gene

PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations

Biotechniques

Discrepancy between molecular structure and ligand selectivity of a testicular FSH receptor of the African catfish (Clarias gariepinus)

Biol. Reprod.

Conversion of human choriogonadotropin into follitropin by protein engineering

Proc. Natl. Acad. Sci. U.S.A.

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein designs

Nat. Biotech.