Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary.

In the intermediate pituitary gland of Xenopus laevis, the expression levels of the prohormone pro-opiomelanocortin (POMC) can be readily manipulated. When the animal is placed on a black background, the gene for POMC is actively transcribed, whereas on a white background the gene is virtually inactive. In this study, we characterized two genes whose transcript levels in the intermediate pituitary are regulated in coordination with that for POMC. One of these codes for a protein homologous to translocon-associated protein TRAP delta, a subunit of a transmembrane protein complex located at the site where nascent secretory proteins enter the endoplasmic reticulum (ER). Both Xenopus and mice were found to express an alternatively spliced transcript that gives rise to a previously unknown version of the TRAP delta protein. The product of the second gene is a novel and highly conserved protein with structural similarity to glycoprotein gp25L, a constituent of another translocon-associated protein complex. A database search revealed the existence of a novel family of gp25L-related proteins whose members occur throughout the animal kingdom. Together, our data imply that (i) the group of ER proteins surrounding translocating polypeptide chains may be far more complex than previously expected, and (ii) a number of the accessory components of the translocon participate in early steps of prohormone biosynthesis.

Molecular probing of the secretory pathway in peptide hormone-producing cells.

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.

Immunocytochemistry and in situ hybridization of neuropeptide Y in the hypothalamus of Xenopus laevis in relation to background adaptation.

The amphibian Xenopus laevis is able to adapt to a dark background by releasing melanophore-stimulating hormone from the pars intermedia of the pituitary gland. The inhibition of melanophore-stimulating hormone release is accomplished by neuropeptide Y-containing axons innervating the pars intermedia. To determine the production site of neuropeptide Y involved in this inhibitory control, the distribution of neuropeptide Y in the brain has been investigated by immunocytochemistry and in situ hybridization. Immunoreactive cell bodies were visualized in, among others, the ventromedial and posterior thalamic nuclei, and the suprachiasmatic and ventral infundibular hypothalamic nuclei. A positive hybridization signal with a Xenopus-specific probe for preproneuropeptide Y-RNA was found in the diencephalic ventromedial thalamic nucleus and in the suprachiasmatic nucleus. With both immunocytochemistry and in situ hybridization, suprachiasmatic neurons appeared to be stained only in animals adapted to a white background; animals adapted to a black background showed no staining. Quantitative image analysis revealed that this effect of background adaptation is specific for suprachiasmatic neurons because no effect could be demonstrated of the background light condition on the ventral infundibular nucleus (immunocytochemistry) or the ventromedial thalamic nucleus (in situ hybridization). These results indicate that neurons in the suprachiasmatic nucleus enable the adaptation of X. laevis to a white background, by producing and releasing neuropeptide Y that inhibits the release of melanophore-stimulating hormone from the melanotrope cells in the pars intermedia of the pituitary gland.

Expression of the Xenopus D2 dopamine receptor. Tissue-specific regulation and two transcriptionally active genes but no evidence for alternative splicing.

In the amphibian Xenopus laevis the D2 dopamine receptor is involved in the regulation of the melanotrope cells of the intermediate pituitary during background adaptation of the animal. The Xenopus D2 receptor has been found to be pharmacologically different from the mammalian D2 receptor. In a number of mammalian species alternative splicing generates two molecular forms of the D2 receptor. These isoforms differ by the presence or absence of 29 amino acids in the third cytoplasmic loop which is thought to be involved in guanine-nucleotide-binding-regulatory-protein (G-protein) binding of the receptor. We previously described a cDNA encoding the large isoform of the Xenopus D2 receptor. Here we report on the isolation of a brain cDNA encoding a second, structurally different Xenopus D2 dopamine receptor. Both Xenopus receptors correspond to the large isoform of the D2 receptor and they display a high degree of sequence identity with their mammalian counterparts. Their occurrence reflects the expression of two Xenopus D2 receptor genes and they are expressed to approximately the same level. In contrast to mammals, PCR analysis gave no evidence for alternative splicing during D2 receptor expression in Xenopus brain and pituitary. Tissue-specific expression of the Xenopus D2 receptor was observed in the pituitary during background adaptation. The low level of receptor mRNA in melanotrope cells of white animals compared to that of black animals may be caused by chronic dopamine stimulation of melanotrope cells in white animals with consequent cellular desensitization and down regulation of the D2 receptor gene.

Cloning and sequence analysis of hypothalamic cDNA encoding Xenopus preproneuropeptide Y.

Neuropeptide Y (NPY) consists of 36 amino acids and it constitutes one of the most conserved neuropeptides. Here we report the complete sequence of the first amphibian NPY precursor by cloning of a hypothalamic cDNA encoding Xenopus laevis preproNPY. The overall amino acid sequence identity between Xenopus and other known NPY precursor proteins ranges from 59% (fish) to 82% (chicken); a low degree of identity was found for the signal peptide sequence (32-75%) and for the carboxy-terminal peptide of NPY (CPON; 43-73%), while the NPY peptide sequence itself constitutes the most highly-conserved region (89-100%) within the preproNPY structure.

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.

Cloning and sequence analysis of pituitary cDNA encoding the beta-subunit of Xenopus proteasome.

The proteasome is a multicatalytic proteinase composed of a number of non-identical subunits. A Xenopus pituitary cDNA was isolated and found to code for the beta-subunit of proteasome. The amino acid sequence deduced from the open reading frame consisted of 215 amino acid residues with a calculated molecular weight of 23,979. A comparative structural analysis indicated that the proteasome subunits can be divided into two groups with the same evolutionary origin. One group consists of subunits with an N-terminally blocked residue and includes components C2, C3, C8 and C9, while the second group of non-blocked proteins includes component C5 and the beta-subunit.