Protein 7B2 Research Articles

The Secretory Granule Protein 7B2 is Secreted in Parallel with Proopiomelanocortin and its End‐Products by the Mouse Corticotroph Tumour Cells AtT‐20

First isolated in porcine pituitary glands the protein 7B2 subsequently proved to be a specific biochemical marker of the secretory granules. Likewise 7B2 was detected in almost all normal and tumoral endocrine tissues. Unexpectedly, several authors failed to demonstrate its presence in rat and human adrenocorticotrophin (ACTH)-secreting cells. In order to definitely establish whether this cell type also produces 7B2 we chose the mouse pituitary corticotroph tumour cell line AtT-20 as a model. Serial dilutions of the mouse culture medium generated displacement curves parallel to that of the standard in a specific 7B2 RIA directed against the human 7B2(23-39) fragment. Under basal secretory conditions immunoreactive 7B2 accumulated in the culture medium in parallel with proopiomelanocortin (POMC) and its fragments N-terminal-joining peptide (NT-JP), joining peptide (JP), beta-lipotropin (beta-LPH), and beta-endorphin (beta-end), although at a much lower (approximately 100-fold) molar concentration. As expected mouse corticotroph cells responded to the stimulatory action of cyclic AMP (3.5 mM) with a preferential increase in the release of POMC end-products, JP and beta-end, which was accompanied by a parallel increase in immunoreactive 7B2 release.

Coordinated expression of 7B2 and αMSH in the melanotrope cells of Xenopus laevis

7B2 is a highly conserved protein present in many secretory cells. Using in situ hybridization techniques and immunocytochemistry, parameters concerning the biosynthesis and storage of the 7B2 protein were studied in the pituitary gland and median eminence of the clawed toad Xenopus laevis, in relation to the physiological process of background adaptation. 7B2-like immunoreactivity was present in the median eminence, in the neural and anterior pituitary lobes and, particularly, in the melanotrope cells of the intermediate pituitary lobe. In these cells, it coexisted with immunoreactivity to proopiomelanocortin (POMC)-derived alpha-melanocyte stimulating hormone (alpha MSH). The melanotropes of black-adapted animals had abundant 7B2-mRNA and POMC-mRNA; melanotropes of white-adapted toads had only low levels of these mRNAs. The presence of 7B2 in nerve terminals and endocrine cells supports the idea that the protein has a general function in the cellular secretory process. In X. laevis, 7B2 appears to be particularly associated with POMC and alpha MSH and, therefore, may play a role in the regulation of background adaptation.

Glucagonomas of transgenic mice express a wide range of general neuroendocrine markers and bioactive peptides

Pancreatic tumours of transgenic mice carrying a glucagon-promoted simian virus 40 (SV40) T antigen oncogene have been analysed by histological, histochemical, ultrastructural and radioimmunological means. Seven transgenic mice were examined revealing dysplastic and neoplastic lesions in the endocrine pancreas. Four tumours were identified, one of which metastasized to periadrenal spaces and paravertebral lymph nodes. Benign tumours were composed of argyrophilic, endocrine cells reactive to a range of antibodies against neuroendocrine markers (neuron-specific enolase, protein gene product 9.5, chromogranin A, synaptophysin and protein 7B2) and different fragments of the proglucagon molecule (glucagon, glicentin, glucagon-like polypeptides 1 and 2). A few tumour cells expressed pancreatic polypeptide, somatostatin or insulin. Conventional ultrastructural analysis and immunogold labelling revealed typical glucagon-immunoreactive alpha granules which co-stored glicentin and glucagon-like polypeptides 1 and 2. The malignant primary tumour and its metastases were composed mainly of cells which did not show immunoreactivity for neuroendocrine markers or peptides. Atypical, glucagon-immunogold labelled granules were detected at electron microscopy in differentiated tumour cells and C-type retroviral particles in the largest tumour population of degranulated cells. The transgene-encoded oncoprotein SV40 large T-antigen was detected in the nuclei of well-differentiated tumour cells and in alpha cells of some dysplastic islets. All tumour-bearing mice showed high levels of circulating glucagon-like immunoreactivity. Transgenic mice harbouring the glucagon-promoted SV40 T antigen oncogene may provide a model for human glucagonoma.

The messenger RNA for pituitary protein 7B2 is widely expressed in mouse CNS and castration reduces its pituitary level

Abstract Mouse organs were examined for the presence of mRNA encoding the pituitary protein 7B2 using Northern blot and quantitative slot blot analysis. The mRNA was most abundant not only in the pituitary (312±12 pg/μg total RNA) as anticipated, but also in many regions of the brain (166±21 pg/μg total RNA overall). The concentration of 7B2 transcripts in different brain areas varied less than four-fold; it was highest in hypothalamic nuclei and lowest in the nucleus accumbens. The relative level of immunoreactive 7B2 among organs often showed no direct correlation with that of 7B2 mRNA, suggesting variable turnover rate of these macromolecules in different tissues. The protein was accumulated in the pituitary more than in any other organ. Several peripheral organs, such as the kidney, the spleen and the lung, previously thought not to express this gene also contained detectable, albeit lower levels of 7B2 mRNA and protein. Orchidectomy significantly reduced 7B2 mRNA level in the pituitary, even in the presence supplementary testosterone. Pending the elucidation of the cellular function of the 7B2, the biological significance of the elevated content of its mRNA in the brain remains uncertain.

The production by alternate splicing of two mRNAs differing by one codon could be an intrinsic property of neuroendocrine protein 7B2 gene expression in man

Two types of mRNAs for neuroendocrine protein 7B2 were deduced from the sequence of cDNAs clones isolated from a human pituitary cDNA library. One type lacks an Ala 100 codon present in the other. The difference is located at an intron site within the human 7B2 gene and can be explained by the transcriptional utilization of two alternate acceptor splice sites, three nucleotides apart. Heteroduplex analysis of DNA fragments amplified by the polymerase chain reaction indicated that this 7B2 mRNA dimorphism occurs in several human endocrine tissues as well as in other species, suggesting that the alternate processing of 7B2 gene transcripts may be an intrinsic mechanism of its expression and could underlie some yet unknown biological functions.

The neuroendocrine polypeptide 7B2 is a precursor protein.

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.

Assignment of the gene for neuroendocrine protein 7B2 ( SGNE1 locus) to mouse chromosome region 2[E3–F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3–F3 region of mouse chromosome 2 and to the q11–q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11–15q13 region.

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein

Mouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of M r 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.

Elevation of a novel pituitary protein (7B2) in the plasma in small cell carcinoma of the lung

7B 2 is a new protein isolated from human pituitary glands and distributed widely, particularly with high concentrations in the neuroendocrine tissues in the rat. We measured plasma 7B 2 concentrations in 333 normal subjects, 20 patients with benign pulmonary disease and 111 patients with primary lung cancer ( 21 small cell carcinoma, 90 non-small cell carcinoma). A normal range for plasma 7B 2 concentrations was defined as less than the mean + 3 S.D. value ( 110 pg/ml) based on plasma 7B 2 concentrations in 333 normal subjects. Elevation of the plasma 7B 2 concentration over the normal range was observed in 15 of 21 patients ( 71.4%) with small cell carcinoma, eight of 90 ( 8.9%) with non-small cell carcinoma and four of 20 ( 20%) with benign pulmonary disease. Plasma 7B 2 concentrations correlated with the clinical course on chemotherapy in some patients with small cell carcinoma. Immunocytochemical studies revealed numerous 7B 2-positive cells in the small cell carcinoma specimen, while 7B 2 staining was not observed in the non-small cell carcinoma and the normal lung specimens. These findings suggest that 7B 2 is secreted by the small cell carcinoma of the lung, which caused elevation of plasma 7B 2 in these patients. 7B 2 might be a possible plasma tumor marker for the small cell carcinoma of the lung.

A new pituitary protein 7B2 is increased in patients with high α- or β-hCG

The presence of 7B2 (a 180 amino acid peptide first extracted from the porcine pituitary) was investigated by a specific radioimmunoassay and gel filtration chromatography in the plasma of three groups of patients known to have increased levels of alpha- and beta-hCG subunits. Plasma 7B2 immunoreactive equivalents (7B2-IE) were increased in postmenopausal women (range 67-143 pmol/l, median: 94 pmol/l, N = 20, P less than 0.05), in patients with Klinefelter's syndrome (range 195-230 pmol/l), median 213 pmol/l, N = 4, P less than 0.01) and in 17 patients with malignant endocrine gastrointestinal tumours (range 66-20,000 pmol/l, N = 32) compared with healthy controls (range 23.6-98.2 pmol/l, median 41.5 pmol/l, N = 40). Tumour tissue from 4 patients with endocrine pancreatic tumours had significantly higher 7B2-IE concentrations than normal pancreatic tissue, with the highest concentration in an insulinoma (449 pmol/g, normal: 28 pmol/g). During therapy almost parallel changes in plasma 7B2 and other hormones were noted and 7B2-IE therefore might be an additional marker for cancers of the APUD system.

CDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice

The cDNA for a widely distributed neuroendocrine protein called 7B2 has been cloned from beta cell tumors of transgenic mice and sequenced. As deduced from the cDNA sequence, 7B2 is a secretory protein of 186 amino acids, nearly identical to its human and porcine homologs. The presence of several pairs of basic residues in the carboxyl terminal portion of the protein suggests that 7B2 can undergo proteolytic maturation in secretory granules and thus generate potential bioactive peptides. 7B2 mRNA is about 1.5 kilobase long and is apparently transcribed from a single gene per haploid genome. The use of tissue-specific promoters to express oncogenes in rare cell types of transgenic mice is a powerful tool for immortalization and expansion of these cells, and it facilitates the isolation and the study of rare proteins such as 7B2.

Secretory protein 7B2 is associated with pancreatic hormones within normal islets and some experimentally induced tumors.

7B2 is a novel neuroendocrine polypeptide which belongs to an entirely new superfamily of proteins. In extension of previous reports on 7B2, these studies concern its expression in endocrine pancreatic tissue. They have been performed using specific antibodies prepared against two distinct synthetic fragments of 7B2 comprising amino acids 23-39 and 117-128 of the native human molecule isolated from pituitary gland. Pancreatic insulin-secreting tumors produced in transgenic mice contain high amounts of 21,500- to 22,000-dalton forms of 7B2. Using light microscopy (immunocytochemical colocalization with different pancreatic hormones), immunoreactivity to 7B2 (IR-7B2) was consistently found within cells producing insulin and glucagon and less consistently within pancreatic polypeptide-containing cells. As in previous reports concerning the brain, adenohypophysis, and thyroid gland, IR-7B2 could be detected by electron microscopy within secretory granules of alpha- and beta-like cells in islets. Furthermore, the IR-7B2 level was higher in extracts of insulin-producing tumors of the transgenic mice that contained the hybrid insulin II gene. In addition, IR-7B2 could be detected immunocytochemically in three of seven tumors produced in the rat by streptozotocin-nicotinamide treatment.

Identification and localization of 7B2 protein in human, porcine, and rat thyroid gland and in human medullary carcinoma.

The novel, highly conserved polypeptide 7B2, which belongs to a new protein superfamily, was isolated from human and porcine hypophysis. The availability of a specific antibody to a synthetic fragment enabled 7B2 localization in a number of neurocrine and endocrine tissues and revealed its secretory character. 7B2 was purified from thyroid homogenates by HPLC chromatography and characterized by gel permeation chromatography (dimeric mol wt, approximately 40,000) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (monomeric mol wt, 20,750). By immunocytochemistry 7B2 was colocalized with calcitonin in parafollicular cells and identified within secretory granules by electron microscopy. Three of nineteen human medullary carcinoma cases showed immunoreactive 7B2 within the early and late hyperplasia stages and neoplasia. Results suggest that 7B2 may play a role in endocrine function, possibly as a secretory substance, and may be a histochemical marker in addition to calcitonin for medullary carcinoma.

Distribution of a novel pituitary protein (7B2) in mammalian gastrointestinal tract and pancreas.

The distribution of a novel pituitary protein (7B2) was determined in the gastrointestinal tract and pancreas of four mammalian species (man, pig, guinea pig, and rat) by a specific radioimmunoassay. The highest concentrations of cross-reacting immunoreactive 7B2 (IR-7B2) were observed in the pancreas and the proximal gut (antrum or duodenum). While the intestinal concentrations varied widely among species, pancreatic IR-7B2 concentrations appeared to be similar in all four species. In the rat, pancreatic islets were found to contain high concentrations of IR-7B2 (5.73 +/- 0.14 fmol/islet, mean +/- SEM). Neonatal capsaicin treatment and enteric nerve section did not affect the concentrations of IR-7B2 in the rat intestine. Layer separation of human gut showed that IR-7B2 is mainly (71 +/- 8%) present in the epithelial fraction. Chromatographic analysis of intestinal and pancreatic extracts from the four species on Sephadex G-100 showed the presence of two immunoreactive peaks at Kav 0.3 and 0.6, but there were both inter- and intraspecies variations in the proportions of the larger and smaller molecular forms.

Developmental changes in immunoreactive content of novel pituitary protein 7B2 in human pancreas and its identification in pancreatic tumors.

Developmental changes in concentration of a novel pituitary protein (7B2) were studied immunochemically in the human pancreas from 20 wk of gestation to 4 mo after birth. The concentrations of 7B2 in pancreatic islet tumors and in nesidioblastosis were investigated also. Significant quantities of 7B2-like immunoreactivity (IR-7B2) were found in all developmental stages studied. The highest concentrations of IR-7B2 were found at term [215.4 +/- 40.0 vs. 28.3 +/- 4.4 pmol/g (adult levels), P less than .001]. A high incidence of elevated IR-7B2 concentration in pancreatic islet tumors and nesidioblastosis was found (10 of 12 insulinomas, 5 of 8 glucagonomas, and in all 3 pancreases with nesidioblastosis). Gel-permeation chromatography on Sephadex G-100 showed two immunoreactive peaks in all extracts studied. The main peak (Kav 0.30) of IR-7B2 corresponded to that found in the porcine pituitary gland. The high incidence of elevated IR-7B2 concentrations in pancreatic islet tumors and the increase in IR-7B2 concentrations in the term pancreas and particularly in nesidioblastosis suggest that the novel protein 7B2 may serve as an islet marker.

Developmental changes in immunoreactive content of novel pituitary protein 7B2 in human pancreas and its identification in pancreatic tumors

Developmental changes in immunoreactive content of novel pituitary protein 7B2 in human pancreas and its identification in pancreatic tumors

Evidence for a novel pituitary protein (7B2) in human brain, cerebrospinal fluid and plasma: Brain concentrations in controls and patients with Alzheimer's disease

A novel pituitary protein, designated as 7B2, recently purified in our laboratory was measured using a specific radioimmunoassay in conjunction with immuno-affinity extraction, in cerebrospinal fluid (CSF) and in plasma obtained from normal volunteers. The mean concentrations of immunoreactive (IR)-7B2 were 2154 pg/ml in CSF and 29 pg/ml in plasma. Studies by SDS-poly-acrylamide gel electrophoresis revealed that both CSF IR-7B2 and plasma IR-7B2 have an apparent molecular weight of around 20,000–21,000 as previously observed in various rat tissues. IR-7B2 was also measured in various brain regions obtained from control subjects and patients with Alzheimer's disease. IR-7B2 was widely distributed in the human brain, with the highest concentrations in substantia nigra and caudate. IR-7B2 brain concentrations were found to be similar between control subjects and patients with Alzheimer's disease. Gel permeation chromatography of extracts of various brain regions revealed two major peaks with apparent molecular weights of 45,000–50,000 and 11,000–16,000 in hypothalamus, caudate, frontal cortex, hippocampus, putamen and locus coeruleus, and only one peak with an apparent molecular weight of 14,000–16,000 in substantia nigra and globus pallidus. These data suggest that this novel pituitary protein may play a role of consequence perhaps as a neurotransmitter or as a neuromodulator in the human central nervous system.

Production of pituitary protein 7B2 immunoreactivity by endocrine tumors and its possible diagnostic value.

7B2 is a protein originally isolated from pituitary, which has been shown to be present in the central nervous system and in certain peripheral tissues, with very high concentrations in pancreatic islets. Endocrine and nonendocrine tumors from 185 patients were investigated by RIA for the presence of immunoreactive pituitary protein 7B2. The highest mean concentration of 7B2 immunoreactivity was found in insulinomas [452 +/- 174 (+/- SEM) pmol/g wet wt tissue; n = 16], which was significantly higher than the concentration in normal adult pancreatic tissue (28.3 +/- 4.4 pmol/g; n = 7). High concentrations of 7B2 immunoreactivity also were found in other endocrine tumors. The cellular localization of 7B2 was studied in normal pancreas, pancreas with hyperplastic islets, and endocrine tumors. 7B2 immunoreactivity was localized to B-cells in the normal pancreas and to variable proportions of cells in islet cell hyperplasias, B-cell tumors, and pheochromocytomas. Plasma concentrations of 7B2 immunoreactivity also were determined in 255 patients with established diagnoses of endocrine or nonendocrine tumors. The proportion of patients with elevated plasma concentrations (arbitrarily set at more than 4 SD above the mean) were 42 of 72 with pancreatic islet cell tumors, 7 of 11 with midgut carcinoid tumors, and 5 of 13 with medullary carcinomas of the thyroid. Especially high values were found in patients with glucagonomas (14 of 20), vipomas (12 of 13), and pancreatic polypeptide-producing tumors (5 of 6). Thus, 7B2 immunoreactivity is produced by a variety of different tumors and may serve as a tumor marker, especially in patients with certain pancreatic islet tumors.

A novel pituitary protein (7B2)-like immunoreactivity is secreted by a rat phaeochromocytoma cell line (PC12).

High concentrations of a novel pituitary protein (7B2) have been shown to be present in the PC12 rat phaeochromocytoma cell line by radioimmunoassay. 7B2-like immunoreactivity (IR-7B2) was released from PC12 cells into the incubation medium in response to stimulation by a depolarizing concentration of K+, and this K+-evoked release was inhibited by Co2+. The major IR-7B2 in PC12 cell and medium appeared to be identical to that in porcine pituitary gland as judged by both gel permeation chromatography and by reverse-phase high performance liquid chromatography (HPLC). Gel permeation chromatography of extracts of cell and medium revealed two IR-7B2 peaks, the earlier eluting at a elution coefficient (Kav) of 0.30 and the later at a Kav of 0.54. In medium, over 90% of the IR-7B2 eluted as the earlier peak. Fractionation of extracts of cell and medium on reverse-phase HPLC showed three main IR-7B2 peaks eluting at 43, 44.5 and 46% acetonitrile/water with 0.1% trifluoroacetic acid. The findings suggest that IR-7B2 might be released by calcium-mediated exocytosis.

Immunocytochemical localization of a novel pituitary protein (7B2) within the rat brain and hypophysis.

A novel pituitary protein called 7B2 was localized in rat pituitary and brain by immunocytochemistry (unlabeled antibody technique). Immunoreactive material was present in the secretory cells of anterior and intermediate lobes and in neural structures of the posterior lobe of the hypophysis. 7B2-immunoreactive neurons were evident within the hypothalamus in the supraoptic nucleus, paraventricular nucleus (magnocellular and parvocellular parts), and lateral hypothalamus. Immunoreactive nerve fibers were seen within the internal and external zone of the median eminence. Among extrahypothalamic regions, the substantia nigra, dorsal tegmental nucleus, cuneiform nucleus, dorsal parabrachial nucleus, spinal tract trigeminal nerve, interior olive, solitary nucleus, and layers I and II of the spinal cord contained 7B2-immunoreactive material. This anatomical distribution suggests a role for 7B2 in endocrine and autonomic functions.