Mammalian Glycoprotein Hormone Research Articles

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): Insights into the evolution of thyrotropin receptor specificity

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH–TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T4-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone–receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11–20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin β subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T4, thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.

Identification of a luteinizing hormone-selective determinant in the exodomain of a follicle-stimulating hormone receptor

Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognate hormones. In contrast, the follicle-stimulating hormone receptor of the African catfish (cfFSHR) is promiscuously activated by catfish luteinizing hormone (cfLH). Glycoprotein hormones bind to the concave site of the cusp-shaped N-terminal GpHR exodomain, which is formed by 9–10 parallel β-strands. Hence, hormone selectivity of each GpHR for its cognate ligand is defined by amino acid sequence divergence in these β-strands between different GpHRs. To identify the molecular determinants that allow promiscuous activation of the cfFSHR by cfLH, β-strands were systematically exchanged between the cfFSHR and the human FSHR. Both gain-of-function and loss-of-function mutational approaches revealed that β-strand 2 of the cfFSHR contains determinants that contribute to the receptor’s responsiveness to cfLH.

Novel Poly-GalNAcβ1–4GlcNAc (LacdiNAc) and Fucosylated Poly-LacdiNAc N-Glycans from Mammalian Cells Expressing β1,4-N-Acetylgalactosaminyltransferase and α1,3-Fucosyltransferase

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.

Characterization of biologically active bovine pituitary FSH purified by immunoaffinity chromatography using a monoclonal antibody

A substantial amount of highly purified, biologically active bovine FSH was isolated from pituitary extracts by immunoaffinity chromatography based on a novel anti-bovine FSH β-subunit monoclonal antibody. The biological activity was assessed in vitro using a steroidogenic granulosa cell line constitutively expressing the FSH receptor. Amino acid analysis, N-terminal amino acid sequencing, and peptide mass mapping demonstrated that primary structure modifications do not contribute to the heterogeneity of bovine FSH. The monosaccharide composition of the N-linked oligosaccharides was quantified and remarkably two distinct forms of sialic acids, N-acetyl- and N-glycolyl-neuraminic acids were found. In conclusion, we showed that isoform differences in bovine FSH is likely due only to sugar chain heterogeneity, and we give the first evidence that two substituted sialic acids contribute to the diversity of mammalian glycoprotein hormone isoforms.

Gonadotropins in insects: an overview.

Control of gonad development in insects requires juvenile hormone, ecdysteroids, and a peptidic brain gonadotropin(s). Compared to vertebrates, the situation in insects with respect to the molecular structure of gonadotropins is far less uniform. Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) of vertebrates are glycoproteins that are synthezised in the hypothalamus and released from the anterior pituitary. They stimulate gonad development, the production of progesterone or of sex steroids (estrogens, androgens). None of the known insect gonadotropins is a glycoprotein, neither can they be grouped into a single peptide family. In Drosophila, two G-protein coupled receptors, structurally related to the mammalian glycoprotein hormone receptors, have been identified. Nothing is known about their natural ligands. The sex-steroids of insects are likely to be ecdysteroids (20E in females, E in males of some species). Some of the identified gonadotropins speed up vitellogenesis (locust OMP and some -PF/-RFamide peptides) or stimulate ecdysteroid production by the ovaries (locust-OMP and Aedes- OEH) or testis (testis ecdysiotropin of Lymantria). In flies, the only as yet identified gonadotropin is the cAMP-generating peptide of Neobellieria. The seeming absence of uniformity in gonadotropins in insects might be due to a multitude of factors that can stimulate ecdysteroid production and/or to the use of different bioassays. Arch.

Differentiation-associated expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides in mammary epithelial cells.

Not only mammalian pituitary glycoprotein hormones but also many glycoproteins from a variety of animal species have been shown to contain N-linked oligosacchardies with the GalNAcbeta1 --> 4GlcNAc structure. Two types of beta-1,4-GalNAcT were found; one transfers N-acetylgalactosamine to acceptor oligosaccharides, which is stimulated by the hormone peptide and the other simply transfers sugar without such activation. In the case of bovine mammary membrane glycoproteins, the expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides was associated with the functional differentiation of the epithelial cells. In contrast, the expression level of such oligosaccharides was much reduced in glycoprotein samples from human breast tumors compared with those from the unaffected regions. These results strongly suggest that the beta-N-acetylgalactosaminylation is one which is regulated under cellular differentiation and dedifferentiation of the mammary gland. Whether or not beta-N-acetylgalactosaminylated N-linked oligosaccharides have unique functions in addition to clearance of the hormone from the circulation remains to be elucidated.

Characterization of two fly LGR (leucine-rich repeat-containing, G protein-coupled receptor) proteins homologous to vertebrate glycoprotein hormone receptors: constitutive activation of wild-type fly LGR1 but not LGR2 in transfected mammalian cells.

The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells.

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.

1-Methyladenine: a starfish oocyte maturation-inducing substance

1-Methyladenine (1-MeAde) in starfish was the first compound to be identified as an oocyte maturation-inducing substance (MIS) among invertebrates in 1969 by Kanatani and co-workers. In starfish, the ripe ovary contains a huge number of fully grown oocytes of almost equal size. Each oocyte possesses a large nucleus (germinal vesicle, GV), which is arrested in late prophase of the first maturation division. The oocyte is surrounded by a single follicle layer. Such immature oocytes are not fertilisable. Resumption of meiosis in immature oocytes can be induced by 1-MeAde, and the mature oocytes thus become fertilisable (Kanatani et al., 1969; Kanatani, 1985). 1-MeAde is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves (Fig. 1).It has been demonstrated that GSS is a peptide hormone (Kanatani et al., 1971). The action of GSS on 1-MeAde production in follicle cells appears to be mediated by its receptor, G-proteins and adenylyl cyclase (Mita & Nagahama, 1991). These findings suggest that a G-protein coupled (seven transmembrane type) receptor is involved in GSS signal transduction, similarly to the pituitary-gonadal axis in vertebrates.Thus, using degenerate probes derived from consensus sequences of the mammalian glycoprotein hormone (GTH and TSH) receptors, cDNA was cloned from mRNA of ovaries of Asterina pectinifera. The cDNA showed striking structural homology with members of the glycoprotein hormone receptor family in the transmembrane region, and contained a very large extracellular region. Expression was observed in isolated ovarian follicle cells. Thus, it seems likely that the glycoprotein hormone receptor (GTHR) family gene is related to GSS receptor in ovarian follicle cells. The phylogenic relatedness of the starfish GTHR was also assessed in relation to other vertebrate GTHRs. The analysis showed that the starfish gene diverged before differentiation of the gonadotropin (LH and FSH) and TSH receptors in vertebrates.

Genomic Organization of a Receptor from Sea Anemones, Structurally and Evolutionarily Related to Glycoprotein Hormone Receptors from Mammals

Cnidarians (e.g., sea anemones and corals) are the lowest animal group having a nervous system. Previously, we cloned a receptor from sea anemones that showed a strong structural similarity to the glycoprotein hormone (TSH, FSH, LH/CG) receptors from mammals. Here, we determine the genomic organization of this sea anemone receptor. The receptor gene contains eight introns that are all localized within a region coding for the large extracellular N terminus. These introns occur at the same positions and have the same intron phasing as eight introns in the genes coding for the mammalian glycoprotein hormone receptors, indicating that the cnidarian and mammalian receptor genes are evolutionarily related. As with the mammalian receptor genes, the sea anemone receptor gene does not contain introns in the region coding for the transmembrane and intracellular domains. Southern blot analyses show that the cnidarian receptor is coded for by a single gene.

Molecular Cloning, Genomic Organization, and Developmental Regulation of a Novel Receptor from Drosophila melanogaster Structurally Related to Members of the Thyroid-stimulating Hormone, Follicle-stimulating Hormone, Luteinizing Hormone/Choriogonadotropin Receptor Family from Mammals

Using oligonucleotide probes derived from consensus sequences for glycoprotein hormone receptors, we have cloned an 831-amino acid residue-long receptor from Drosophila melanogaster that shows a striking structural homology with members of the glycoprotein hormone (thyroid-stimulating hormone (TSH); follicle-stimulating hormone (FSH); luteinizing hormone/choriogonadotropin (LH/CG)) receptor family from mammals. This homology includes a very large, extracellular N terminus (20% sequence identity with rat TSH, 19% with rat FSH, and 20% with the rat LH/CG receptor) and a seven-transmembrane region (53% sequence identity with rat TSH, 50% with rat FSH, and 52% with the rat LH/CG receptor). The Drosophila receptor gene is >7.5 kilobase pairs long and contains 17 exons and 16 introns. Seven intron positions coincide with introns in the mammalian glycoprotein hormone receptor genes and have the same intron phasing. This indicates that the Drosophila receptor is evolutionarily related to the mammalian receptors. The Drosophila receptor gene is located at position 90C on the right arm of the third chromosome. The receptor is strongly expressed starting 8-16 h after oviposition, and the expression stays high until after pupation. Adult male flies express high levels of receptor mRNA, but female flies express about 6 times less. The expression pattern in embryos and larvae suggests that the receptor is involved in insect development. This is the first report on the molecular cloning of a glycoprotein hormone receptor family member from insects.

Precise determination of TSH receptor antibody activity in serum containing bovine TSH (bTSH) binding antibody by absorption using denatured bTSH or sheep FSH.

A previous report demonstrated that sera with bovine TSH (bTSH) binding antibody showed abnormally negative TSH receptor antibody (TRAb) activity in the standard TRAb assay method. The corrected TRAb activity calculated by the determination of the nonspecific binding of the labeled bTSH for each test serum [NSB(T)] resulted in positive TRAb activity. However, the precise calculation was difficult because NSB(T) level was significantly higher than the nonspecific binding in normal pool serum [NSB(N)] level. In the present experiment the determination of the TRAb activity was performed after absorption of bTSH binding antibodies by the heat-denatured bTSH to obtain more precise TRAb activity. In addition, absorption by sheep FSH (sFSH) was performed because almost all bTSH binding antibodies showed specific binding to the alpha-subunit of mammalian pituitary glycoprotein hormones in our previous study. Three days absorption of test serum using 1 mU of the heat-denatured bTSH (100 degrees C for 1 h) or 5 mU of sFSH was chosen as optimal because NSB(T) decreased remarkably to NSB(N) levels. The corrected TRAb determined after these absorptions decreased significantly compared to the corrected TRAb activity without the absorption. When the complete absorption of bTSH binding antibody was performed by the decrease of the NSB(T) level to the NSB(N) level, the TRAb activity determined by these two different absorptions was almost similar (difference was less than 10%). However, it was difficult to obtain the precise TRAb activity in the cases with extremely high bTSH binding antibody, because the NSB(T) level was higher than the NSB(N) level by the incomplete absorption of bTSH binding antibody.

Evolutionary Conservation of the Sulfated Oligosaccharides on Vertebrate Glycoprotein Hormones That Control Circulatory Half-life

The circulatory half-life of the mammalian glycoprotein hormone lutropin is controlled by its unique Asn-linked oligosaccharides, which terminate with the sequence SO4-4-GalNAc beta 1,4GlcNAc. A cluster of basic amino acids essential for recognition of the alpha subunit by the glycoprotein hormone:N-acetylgalactosaminyltransferase is located within two turns of an alpha helix (Mengeling, B.J., Manzella, S.M., and Baenziger, J.U. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 502-506). The amino acids within this region are virtually invariant in the alpha subunits of all vertebrates, indicating that the recognition determinant utilized by the N-acetylgalactosaminyltransferase has been conserved in species ranging from teleost fish to mammals. We demonstrate that the glycoprotein hormone:N-acetylgalactosaminyltransferase and the N-acetylgalactosamine-4-sulfotransferase responsible for the synthesis of these unique sulfated oligosaccharides are expressed in the pituitaries of vertebrates ranging from teleost fish to mammals. Furthermore, we show that Asn-linked oligosaccharides terminating with SO4-4-GalNAc beta 1,4GlcNAc are present on the alpha and beta subunits of the salmon glycoprotein hormone GTH II. Asn-linked oligosaccharides terminating with SO4-4-GalNAc beta 1,4GlcNAc are unique structural features of the glycoprotein hormones that have been conserved during vertebrate evolution, suggesting they are critical for the expression of hormone biologic activity.

Identification of conserved amino acid residues in the beta subunit of human choriogonadotropin important in holoprotein formation.

The beta subunits of mammalian glycoprotein hormones contain 20 invariant amino acid residues, including 12 Cys residues that form six disulfide bonds, as well as other highly conserved residues. Such stringent conservation suggests an important role for those residues in chain folding, holoprotein formation, or receptor binding/activation, but not receptor specificity. Using site-directed mutagenesis, we have prepared and characterized replacements of 14 conserved non-Cys residues in human choriogonadotropin beta for which there is little, if any, information available: Pro7, Thr32, Tyr37, Thr40, Val56, Tyr59, Phe64, Pro70, Gly71, Ser81, Tyr82, Val84, Ala85, and Ser87. The cDNAs were subcloned into a pRSV mammalian expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Holoprotein formation was assessed by radioimmunoassays, and in vitro competitive binding and steroidogenic assays were used to determine potencies. Our results demonstrated that Tyr37, Thr40, Tyr59, and Ala85 participate directly or indirectly in holoprotein formation, as may Ser81. Replacements of the other conserved amino acid residues resulted in no significant change in subunit assembly or receptor binding/activation as measured, although an Arg at position 81 or 82 reduced secretion. This study has identified several amino acid residues of the beta subunit that are important in alpha binding and has raised interesting questions on the constraints that maintain conservation of amino acid residues during evolution.

A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction.

We have isolated and analyzed a cDNA from the central nervous system of the mollusc Lymnaea stagnalis encoding a putative receptor, which might be a natural hybrid between two different classes of receptor proteins. Preceded by a signal peptide, two types of repeated sequences are present in the N-terminal part of the protein. The first repeat displays a high sequence similarity to the extracellular binding domains of the low density lipoprotein receptor, which binds and internalizes cholesterol-containing apolipoproteins. The second repeat and the C-terminal part of the Lymnaea receptor are very similar to regions of a specific class of guanine nucleotide-binding protein-coupled receptors, the mammalian glycoprotein hormone receptors. The mRNA encoding the receptor is predominantly expressed in a small number of neurons within the central nervous system and to a lesser extent in the heart.

Molecular Cloning of a Novel, Putative G Protein-Coupled Receptor from Sea Anemones Structurally Related to Members of the FSH, TSH, LH/CG Receptor Family from Mammals

Using oligonucleotide probes derived from consensus sequences of known vertebrate and invertebrate G protein-coupled receptors, we have cloned the cDNA for a presumed G protein-coupled receptor from sea anemones. This receptor shows a striking structural homology with members of the glycoprotein hormone (FSH, TSH, LH/CG) receptor family from mammals, including a very large, extracellular N terminus (18-25% sequence identity) and a 7 transmembrane region (44-48% sequence identity). As with the mammalian glycoprotein hormone receptor genes, the sea anemone receptor gene yields transcripts which can be alternatively spliced, thereby yielding a shortened receptor variant only containing the large extracellular (soluble) N terminus. All this is strong evidence that the putative glycoprotein hormone receptor from sea anemones is evolutionarily related to those from mammals. This is the first report showing that a putative glycoprotein hormone receptor occurs in invertebrates.

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin β subunit

The mammalian glycoprotein hormone β subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34–38 of human chorionic gonadotropin β), that is denoted as the ‘CAGY region’. Using site-directed mutagenesis we have replaced Tyr-37 in hCGβ, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH β subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for α subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine α were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCGβ(Phe-37) mutant bound to α essentially to the same extent as hCGβ wild-type, while the hCGβ(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [ 125I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCGβ without any dramatic effect on function. These results indicate that Tyr-37 in hCGβ, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.

Molecular Cloning and Neuronal Expression of a Novel Type of a G-Protein-Coupled Receptor With Ldl Binding Motifs From the Pond Snail Lymnaea Stagnalis

We have isolated and characterized a cDNA encoding a novel type of receptor from the central nervous system (CNS) of the pond snail Lymnaea stagnalis. This receptor appears to be a natural hybrid between two classes of receptor proteins. The N-terminal part of the protein contains two types of repeated sequences; the first displays a high degree of sequence similarity with the extracellular binding domains of the low density lipoprotcin (LDL) receptor, which binds, internalizes, and releases cholesterol containing apolipoproteins. The second type of repeat, and the C-tcrminal part of this receptor arc homologous to specific regions of a class of G-protein-coupled receptors, the mammalian glycoprotein hormone receptor family. The mRNA encoding the receptor is predominantly located in the CNS in a small cluster of 90 neurons within the pedal ganglia, and to a lesser extent in the heart. The discovery of this chimeric receptor and its neuronal expression indicates the presence of a new subclass of G-protein-coupled receptors that may be involved in the transduction of signals carried by lipoproteins into neuronal events via G-proteins.

Primary structure of two mRNAs encoding putative salmon α-subunits of pituitary glycoprotein hormone

1. 1. By random sequence analyses, we isolated from the cDNA library of salmon pituitary glands two clones, the deduced amino acid sequences corresponding to the C-terminal region of which are almost the same as those of the α subunits of mammalian glycoprotein hormones. 2. 2. Comparison of the nucleotide sequences and deduced amino acid sequences from these two clones with those of mammalian species revealed that the two newly-isolated cDNAs corresponded to mRNAs encoding the putative salmon pre-α subunit of glycoprotein hormones. 3. 3. Homology in the nucleotide sequences of these two clones suggested that corresponding mRNAs may be encoded by separate genes which probably evolved from a common ancestral gene.

Immunocytochemical demonstration of pituitary cell types in the teleost Poecilia latipinna, by light and electron microscopy

Using the unlabelled antibody method at the light microscope level, and the immunogold method at the electron microscope level, the distribution of the different adenohypophysial cells was demonstrated in the teleost Poecilia latipinna, by means of antisera to both teleostean and mammalian pituitary hormones and their subunits. Anti-salmon prolactin, but not anti-rat or-ovine prolactin, gave a specific staining of the acidophils of the rostral pars distalis (RPD), while anti-trout growth hormone (GH), but not anti-rat GH, stained similar but always separate cells in the proximal pars distalis (PPD). Antisera to the whole molecules of mammalian glycoprotein hormones stained the entire population of basophils in the PPD, but separate populations of gonadotrophs and thyrotrophs could be discriminated using anti-salmon gonadotrophin and anti-human thyrotrophin β subunit. Antisera to ACTH (1–24) and (11–24) sequences, as well as β-endorphin and met-enkephalin, stained the lead haematoxylin-positive cells of the RPD and pars intermedia (PI), whereas anti-α-MSH stained only the PI cells. Ultrastructural examination showed that these immunoreactivities were present in the same secretory granules, and were always greater in pale granules rather than electron dense granules. In the RPD, blebs of ACTH-immunoreactive cytoplasm were found to protrude through the gaps in the basement membrane into the neurohypophysis. The second “PAS-positive” cell type of the PI showed a strong cross-reaction with anti-salmon gonadotrophin, suggesting that it may produce a glycoprotein chemically related to the gonadotrophin(s).