Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for multiple processes within the eukaryotic cell, including membrane transport and neurotransmitter secretion. How the V-ATPase is regulated, e.g. by an accessory subunit, remains elusive. Here we explored the role of the neuroendocrine V-ATPase accessory subunit Ac45 via its transgenic expression specifically in the Xenopus intermediate pituitary melanotrope cell model. The Ac45-transgene product did not affect the levels of the prohormone proopiomelanocortin nor of V-ATPase subunits, but rather caused an accumulation of the V-ATPase at the plasma membrane. Furthermore, a higher abundance of secretory granules, protrusions of the plasma membrane and an increased Ca(2+)-dependent secretion efficiency were observed in the Ac45-transgenic cells. We conclude that in neuroendocrine cells Ac45 guides the V-ATPase through the secretory pathway, thereby regulating the V-ATPase-mediated process of Ca(2+)-dependent peptide secretion.     

Transgene expression of prion protein induces crinophagy in intermediate pituitary cells

The cellular form of the prion protein (PrP(C)) is a plasma membrane-anchored glycoprotein whose physiological function is poorly understood. Here we report the effect of transgene expression of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP-PrP(C)) specifically in the neuroendocrine intermediate pituitary melanotrope cells of Xenopus laevis. In the transgenic melanotrope cells, the level of the prohormone proopiomelanocortin (POMC) in the secretory pathway was reduced when the cells were (i) exposed for a relatively long time to the transgene product (by physiologically inducing transgene expression), (ii) metabolically stressed, or (iii) forced to produce unfolded POMC. Intriguingly, although the overall ultrastructure was normal, electron microscopy revealed the induction of lysosomes taking up POMC secretory granules (crinophagy) in the transgenic melanotrope cells, likely causing the reduced POMC levels. Together, our results indicate that in neuroendocrine cells transgene expression of PrP(C) affects the functioning of the secretory pathway and induces crinophagy.     

Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.     

The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway.

The vacuolar H+-ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here, we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact approximately 46-kDa Ac45 was found to be N-linked glycosylated to approximately 62 kDa and a minor fraction to approximately 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of approximately 61 and approximately 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa (the deglycosylated form is approximately 23 kDa), and a previously not detected approximately 22-kDa N-terminal cleavage fragment (the deglycosylated form is approximately 20 kDa). Degradation of the N-terminal fragment is rapid, does not occur in lysosomes and is inhibited by brefeldin A. Both the N- and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4-6 h) and takes place in the early secretory pathway, as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur. Together, our results indicate that Ac45 folding and cleavage occur slowly and early in the secretory pathway, and that the cleavage event may be linked to V-ATPase activation.     

Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24alpha, -beta, -gamma, and -delta. We previously identified a member of the p24delta subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis ( approximately 30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24delta member (Xp24delta(2)) and on the identification and characterization of a second member (Xp24delta(1)) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24delta(2). The two p24delta members are ubiquitously expressed, but Xp24delta(2) is neuroendocrine enriched. During black background adaptation, the amount of the Xp24delta(2) protein in the intermediate pituitary was increased approximately 25 times, whereas Xp24delta(1) protein expression was increased only 2.5 times. Furthermore, the level of Xp24delta(2) mRNA was approximately 5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24delta(1) mRNA expression was not induced. Therefore, the expression of Xp24delta(2) specifically correlates with the expression of POMC. Together, our findings suggest that p24delta proteins have a role in selective protein transport in the secretory pathway.     

Evolutionary conservation of the 14-3-3 protein.

The novel family of 14-3-3 proteins may be involved in the regulation of neuronal activity. During our search for proteins coordinately expressed with the prohormone proopiomelanocortin in the melanotrope cells of the Xenopus intermediate pituitary gland, we cloned and sequenced a pituitary cDNA encoding a Xenopus 14-3-3 protein. Alignment of the Xenopus protein with known mammalian, Drosophila and plant 14-3-3 polypeptide and with a mammalian protein kinase C inhibitor protein revealed that the neuron-specific 14-3-3-related proteins are highly conserved (60-88%) throughout eukaryotic evolution.     

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H⁺)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.     

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.     

Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V(0)-sector is involved in Ca(2+)-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V(0)-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca(2+)-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca(2+)-dependent-regulated exocytosis.     

Comparison of the Molecular Forms of the Kex2/Subtilisin-Like Serine Proteases SPC2, SPC3, and Furin in Neuroendocrine Secretory Vesicles Reveals Differences in Carboxyl-Terminus Truncation and Membrane Association

Abstract: The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.     

The Neuroendocrine Proteins Secretogranin II and III Are Regionally Conserved and Coordinately Expressed with Proopiomelanocortin in Xenopus Intermediate Pituitary

Abstract: Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretory granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans -Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15- and 35-fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30-fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family.     

Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesis

The cellular prion protein (PrP(C)) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrP(C) functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrP(C), the newly synthesized GFP-PrP(C)K81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrP(C), the mature GFP-PrP(C)K81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrP(C) at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrP(C)DeltaGPI and GFP-PrP(C)octa mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrP(C) for its correct biosynthesis.     

Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins.

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.     

Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis

In the present study, we examined the amphibian Xenopus laevis as a model for stable transgenesis and in particular targeted transgene protein expression to the melanotrope cells in the intermediate pituitary. For this purpose, we have fused a Xenopus proopiomelanocortin (POMC) gene promoter fragment to the gene encoding the reporter green fluorescent protein (GFP). The transgene was integrated into the Xenopus genome as short concatemers at one to six different integration sites and at a total of one to approximately 20 copies. During early development the POMC gene promoter fragment gave rise to GFP expression in the total prosencephalon, whereas during further development expression became more restricted. In free-swimming stage 40 embryos, GFP was found to be primarily expressed in the melanotrope cells of the intermediate pituitary. Immunohistochemical analysis of cryosections of brains/pituitaries from juvenile transgenic frogs revealed the nearly exclusive expression of GFP in the intermediate pituitary. Metabolic labelling of intermediate and anterior pituitaries showed newly synthesized GFP protein to be indeed primarily expressed in the intermediate pituitary cells. Hence, stable Xenopus transgenesis with the POMC gene promoter is a powerful tool to study the physiological role of proteins in a well-defined neuroendocrine system and close to the in vivo situation.     

Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.     

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.     

Biosynthesis and differential processing of two pools of amyloid-beta precursor protein in a physiologically inducible neuroendocrine cell

The amyloid-beta precursor protein (APP) is linked to Alzheimer's disease through its pathological proteolytic processing in the secretory pathway. Nevertheless, surprisingly little is known about the biosynthesis of endogenous APP. We therefore decided to investigate the intracellular fate of newly synthesized APP in a physiologically inducible neuroendocrine cell, the Xenopus intermediate pituitary melanotrope cell. We found that the level of both APP mRNA and protein was about threefold induced in the activated cells of black-adapted animals. Intriguingly, two pools of APP were found, only one of which was up-regulated. This induced pool became readily N- and subsequently O-glycosylated and was eventually proteolytically processed by an alpha-secretase-like cleavage event resulting in a secreted N-terminal and a cell-associated C-terminal APP fragment. Conversely, only the other (non-induced, non-glycosylated and uncleaved) pool became phosphorylated. Thus, we report on the biosynthesis of APP in a physiological context and illuminate the occurrence of two pools of APP, one of which is linked to neuroendocrine cell activation.     

A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell

The p24alpha, -beta, -gamma, and -delta proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24delta2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24delta2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.     

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.