Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

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.

The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family.

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Evidence for a conformational polymorphism of invertebrate neurohormones. D-amino acid residue in crustacean hyperglycemic peptides.

Several large peptidic neurohormones have been isolated in crustaceans. In lobster and other related species, each of these neurohormones, and particularly the crustacean hyperglycemic hormone, occurs as two isoforms having the same peptidic sequence and molecular mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third residue (Phe3) of the lobster hyperglycemic hormones is in either the L- or D-configuration. In addition, we have shown that the biological activity of the two isoforms differs when considering the kinetics of their hyperglycemic effect.

Localization of messenger RNAs encoding crustacean hyperglycemic hormone and gonad inhibiting hormone in the X-organ sinus gland complex of the lobster Homarus americanus.

The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of crustacean hyperglycemic hormone (CHH) from the crayfish, Orconectes limosus: emergence of a novel neuropeptide family.

The primary structure of the major form of CHH from sinus glands of the crayfish, Orconectes limosus, was determined by manual Edman microsequencing. It is a 72-residue peptide with a calculated Mr of 8400 Da. In the number of residues, it is identical to the CHH of Carcinus maenas and very similar to MIH (moult inhibiting hormone) of Homarus americanus. All three peptides have pGlu as N-terminus in common, and Val-NH2 is the C-terminal residue in Orconectes and Carcinus CHH. Six Cys residues occupy identical position in the three peptides. There is a 61% sequence identity with Carcinus CHH, and an 81% identity with Homarus MIH.

Cloning and sequence analysis of cDNA encoding two crustacean hyperglycemic hormones from the lobster Homarus americanus.

Using the polymerase chain reaction with degenerated oligonucleotides, we have isolated cDNA clones that encode two structurally different (92% identity) crustacean hyperglycemic hormones (CHH) from the lobster Homarus americanus. The deduced amino acid sequences fully agree with previously published data on partial amino acid sequences, amino acid compositions and molecular masses of hyperglycemic peptides in the lobster. A comparative analysis between the deduced primary structure of two lobster CHH and the crab CHH sequence reveals a phylogenetic relationship and allows the prediction of biologically important regions within the structures of these novel neuropeptides.

Comparative characterization of hyperglycemic neuropeptides from the lobster Homarus americanus.

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.