Marion Blomenröhr

Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus)

Abstract A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

Physiological Roles of Gonadotropin-Inhibitory Hormone Signaling in the Control of Mammalian Reproductive Axis: Studies in the NPFF1 Receptor Null Mouse

RF-amide-related peptide-3 (RFRP-3), the mammalian ortholog of the avian gonadotropin-inhibiting hormone (GnIH), operates via the NPFF1 receptor (NPFF1R) to repress the reproductive axis, therefore acting as counterpart of the excitatory RF-amide peptide, kisspeptin (ligand of Gpr54). In addition, RFRP-3 modulates feeding and might contribute to the integrative control of energy homeostasis and reproduction. Yet, the experimental evidence supporting these putative functions is mostly indirect, and the physiological roles of RFRP-3 remain debatable and obscured by the lack of proper analytical tools and models. To circumvent these limitations, we characterize herein the first mouse line with constitutive inactivation of NPFF1R. Ablation of NPFF1R did not compromise fertility; rather, litters from NPFF1R null mice were larger than those from wild-type animals. Pubertal timing was not altered in NPFF1R deficient mice; yet, pre-pubertal knockout (KO) males displayed elevated LH levels, which normalized after puberty. Adult NPFF1R null male mice showed increased Kiss1 expression in the hypothalamic arcuate nucleus, higher serum FSH levels, and enhanced LH responses to GnRH. However, genetic elimination of NPFF1R was unable to reverse the state of hypogonadism caused by the lack of kisspeptin signaling, as revealed by double NPFF1R/Gpr54 KO mice. NPFF1R null mice displayed altered feedback responses to gonadal hormone withdrawal. In addition, metabolic challenges causing gonadotropin suppression, such as short-term fasting and high-fat diet, were less effective in dampening LH secretion in NPFF1R-deficient male mice, suggesting that absence of this inhibitory pathway partially prevented gonadotropin suppression by metabolic stress. Our data are the first to document the impact of elimination of GnIH signaling on reproductive parameters and their modulation by metabolic challenges. Whereas, in keeping with its inhibitory role, the NPFF1R pathway seems dispensable for preserved puberty and fertility, our results surface different alterations due to the lack of GnIH signaling that prominently include changes in the sensitivity to fasting- and obesity-associated hypogonadotropism.

Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor

The gonadotropin-releasing hormone (GnRH) receptor in catfish differs from its mammalian counterparts in showing a very low affinity for the hypothalamic GnRH form [i.e. catfish GnRH (cfGnRH)] and a very high affinity for the highly conserved mesencephalic GnRH, chicken GnRH-II (cGnRH-II). In the present study we investigated the molecular interactions between ligand and receptor involved in determining the ligand selectivity of the catfish GnRH receptor. Studies on the binding characteristics of the catfish GnRH receptor for cfGnRH and cGnRH-II as well as for mammalian GnRH (mGnRH) and synthetic chimaeric GnRHs, differing at positions 5, 7 and 8, revealed that the low affinity of the catfish receptor for cfGnRH can be improved by replacing Leu(7) by a tryptophan residue and/or Asn(8) by either a tyrosine or an arginine residue. Testing cfGnRH and cGnRH-II as well as mGnRH and the chimaeric GnRHs on Asp(304)-->Ala, Asp(304)-->Glu and Asp(304)-->Asn mutant catfish GnRH receptors revealed that Asp(304) of the catfish receptor mediates the recognition of Arg(8) in mGnRH, as well as in the chimaeric peptides [Arg(8)]cfGnRH and [Arg(8)]cGnRH-II, but seems to be less important for the recognition of Tyr(8) in cGnRH-II. On the basis of these results, a three-dimensional model for the binding of [Arg(8)]cGnRH-II to the catfish GnRH receptor is proposed.

Differences in structure—function relations between nonmammalian and mammalian GnRH receptors: what we have learnt from the African catfish GnRH receptor

Publisher Summary Mammalian gonadotropin-releasing hormone (GnRH) receptors differ from other G-protein coupled receptors (GPCRs) in lacking the intracellular C-terminal tail and in showing an exchange of two otherwise highly conserved aspartate protease (Asp) and asparaginyl (Asn) residues in TM 2 and 7, respectively. However, the first GnRH receptor characterized from a nonmammalian vertebrate, the African catfish, contains an intracellular C-terminal tail and has Asp residues. Next to structural variations, differences between mammalian and nonmammalian GnRH receptors are found in their pharmacology and their regulation. In addition, the results of studies on catfish and mammalian GnRH receptor expression, regulation and activation, and ligand binding are summarized. To this end, mammalian-nonmammalian chimeric GnRH receptors, site-directed mutagenesis of GnRH receptors, various GnRH analogs, and a three-dimensional model of the receptor-ligand complex were used. It is demonstrated that the catfish GnRH receptor is susceptible to agonist-induced phosphorylation and that Ser in the C terminal tail is the major phospho-acceptor site in this process. Mammalian GnRH receptors, on the contrary, are resistant to agonist-dependent phosphorylation due to the lack of a C-terminal tail.

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp90 residue

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein‐coupled receptors, except in mammalian gonadotropin‐releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp90 of the catfish GnRH receptor can be substituted by a neutral Asn90 without abolishing receptor signalling if another negatively charged Glu93 is introduced in a proximal region of the receptor interior, thereby mimicking the Glu90–Lys121 salt bridge of mammalian GnRH receptors.

Characterization of neocortical and hippocampal synaptosomes from temporal lobe epilepsy patients.

To investigate epilepsy-associated changes in the presynaptic terminal, we isolated and characterized synaptosomes from biopsies resected during surgical treatment of drug-resistant temporal lobe epilepsy (TLE) patients. Our main findings are: (1) The yield of synaptosomal protein from biopsies of epilepsy patients was about 25% of that from rat brain. Synaptosomal preparations were essentially free of glial contaminations. (2) Synaptosomes from TLE patients and naive rat brain, quickly responded to K(+)-depolarization with a 70% increase in intrasynaptosomal Ca(2+) ([Ca(2+)](i)), and a 40% increase in B-50/GAP-43 phosphorylation. (3) Neocortical and hippocampal synaptosomes from TLE patients contained 20-50% of the glutamate and gamma-aminobutyric acid (GABA) contents of rat cortical synaptosomes. (4) Although the absolute amount of glutamate and GABA released under basal conditions from neocortical synaptosomes of TLE patients was lower than from rat synaptosomes, basal release expressed as percentage of total content was higher (16.4% and 17.3%, respectively) than in rat (11.5% and 9. 9%, respectively). (5) Depolarization-induced glutamate and GABA release from neocortical synaptosomes from TLE patients was smaller than from rat synaptosomes (3.9% and 13.0% vs. 21.9% and 25.0%, respectively). (6) Analysis of breakdown of glial fibrillary acid protein (GFAP) indicates that resection time (anoxic period during the operation) is a critical parameter for the quality of the synaptosomes. We conclude that highly pure and viable synaptosomes can be isolated even from highly sclerotic human epileptic tissue. Our data show that in studies on human synaptosomes it is of critical importance to distinguish methodological (i.e., resection time) from pathology-related abnormalities.

Receptor Mutagenesis Strategies for Examination of Structure-Function Relationships

This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base substitutions/deletions/insertions that yield single/multiple amino acid substitutions/deletions/insertions and/or N- or C-terminal truncations in GPCRs. Polymerase chain reaction-based mutagenesis strategies allow substitutions/deletions/insertions of larger domains within GPCRs, creating truncated receptors or receptor chimeras. In addition, some guidelines are given and examples are provided to facilitate design and interpretation of mutational experiments.