Structural analysis and sheep pituitary receptor binding of GnRH and its complexes with metal ions
Introduction
The gonadotropin-releasing hormone (GnRH) or luteinizing hormone releasing hormone (LHRH) is a deca-peptide (pEHWSYGLRPG) and plays an essential role in mediating the neuro-endocrine control of reproductive processes. In a previous investigation of metal–GnRH complexes interacting with the rat pituitary receptor [1], the copper(II) complex was shown to act more efficiently than native GnRH; whereas the activity of nickel(II) or zinc(II) complexes was slightly lower. Metal complexation was also shown to distinctly affect the ovulation activity of the peptide hormone [2].
Structural features of Ni(II) complexes are herein investigated with the aim of:
- (i)
assessing how metal binding affects the peptide preferred conformation, if any, assumed by GnRH in solution;
- (ii)
delineating the activity of free and metal-bound GnRH with the sheep pituitary receptor;
- (iii)
relating the structures of free and bound GnRH with biological activity.
Studies of GnRH binding Ni(II) have already been performed [3], but precipitation of a solid complex from aqueous solution at pH≥9–10 has impeded investigations of 1:1 mixtures at strongly basic pH. In order to avoid such complications, DMSO was chosen as solvent and sodium methoxide (CH3ONa) was used as a strongly deprotonating Lewis base. In the course of the study, previous results on the ‘preferred’ conformation of free GnRH in solution [4] and on structural features of the nickel complex were ratified and extended [3].
Section snippets
Materials and methods
GnRH was purchased from Sigma and used without further purification. Ni(ClO4)2·6H2O, Cu(ClO4)2·6H2O, and Co(ClO4)2·6H2O were purchased from Alfa Aesar. Dimethyl-d6 sulfoxide (DMSO-d6 100%) and sodium methoxide (100%) were also obtained from Sigma.
NMR experiments were carried out on a Bruker AMX-600 spectrometer at 298±0.2 K. The GnRH solution was 6.4 mM in DMSO-d6 and it was carefully deoxygenated by repeated freezing–thawing cycles. Chemical shifts were referenced to TMS (tetramethylsilane).
Results and discussion
The results obtained from binding studies to the sheep pituitary receptor are reported in Fig. 1. The GnRH agonist buserelin (d-Ser(tBu)6-Pro9-NHEt)-GnRH is shown to compete with GnRH for specific binding sites on the receptor (Fig. 1a). When comparing metal–GnRH complexes with free GnRH (Fig. 1b, c) it is found that, while the copper(II) complex is more effective than the free hormone, the nickel(II) and cobalt(II) complexes display a lower binding affinity. These findings ratify and extend
Conclusions
The interaction of GnRH with its receptor is directly affected by Cu(II), Ni(II) and Co(II). The metal can act either as agonist [Cu(II)] or antagonist of the interaction [Ni(II) and Co(II)]. The NMR study on the free ligand reveals the occurrence of two structured ‘domains’ in the peptide, the relative orientation of which is modulated by a mobility of the central glycine. This degree of freedom may help in accommodating the peptide at the receptor site. The structure of the Ni(II)–GnRH
References (17)
- et al.
J. Inorg. Biochem.
(1997) - et al.
J. Inorg. Biochem.
(1990) - et al.
J. Inorg. Biochem.
(1988) - et al.
J. Mol. Biol.
(1997) - et al.
J. Magn. Reson. A
(1996) - et al.
Biochem. Biophys. Res. Commun.
(1975) - et al.
Magn. Reson. Chem.
(1994) J. Am. Chem. Soc.
(1976)
Cited by (15)
Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B
2020, Biochemical and Biophysical Research CommunicationsGonadotropin-releasing hormone-Cu complex (Cu-GnRH) transcriptional activity in vivo in the female rat anterior pituitary gland
2020, Brain Research BulletinCitation Excerpt :In this aspect, it may be considered a unique GnRH analogue which, relative to GnRH, more potently stimulates LH release in ovariectomized rats (Kochman et al., 1992) and in gonadotrope cells primary culture (Michaluk et al., 2006). Cu-complex binds to GnRH receptors with greater affinity (Kochman et al., 1997; D’Amelio et al., 2003) and its reduced susceptibility to proteolytic cleavage due to rendered access of the specific endopeptidases to AA bond (Herman et al., 2012) may also contribute to the extension of ligand availability and in consequence to act in favor to prolong Cu-GnRH-induced receptor activation. Furthermore, studies on intracellular signal transduction revealed the ability of the complex to activate cAMP/PKA signalling in porcine (Kochman et al., 2005) and rat anterior pituitary primary culture (Gajewska et al., 2016), whereas non–complexed GnRH failed to do so.
Neurokinin B and serum albumin limit copper binding to mammalian gonadotropin releasing hormone
2018, Biochemical and Biophysical Research CommunicationsImpact of the Cu(II) ions on the chemical and biological properties of goserelin – coordination pattern, DNA degradation, oxidative reactivity and in vitro cytotoxicity
2017, Journal of Inorganic BiochemistryCitation Excerpt :Therefore, a novel modulatory role of GnRH in the context of tumors growth promotion, metastasis and angiogenesis has been reported [13–15]. Treatment of the hormone-dependent tumors is connected with the desensitization of the pituitary which suppress the secretion of GnRH and sex steroids, leading to pharmacological castration [15,16]. Comprehensive studies have indicated that metal ions may affect the endocrine activity of GnRH [16].
Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro
2016, Brain Research BulletinCitation Excerpt :Existing data suggest that specific properties of the Cu-GnRH molecule might act to prolong GnRH-R stimulation and thus contribute to enhanced cAMP synthesis. Indeed, Cu-GnRH interacts with GnRH receptors with greater affinity than GnRH (D’Amelio et al., 2003), but is also less susceptible to proteolytic degradation (Herman et al., 2012). Cu-GnRH-induced cAMP levels were observed in the presence of IBMX, a potent phosphodiesterase inhibitor, indicative of activation of AC rather than inhibition of cAMP degradation.