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B A Eipper

Publications and source records attributed to B A Eipper.

At least 55 records · Page 3Linked to original sources
PubMed1997

Neuropeptide amidation in Drosophila: separate genes encode the two enzymes catalyzing amidation.

In vertebrates, the two-step peptide alpha-amidation reaction is catalyzed sequentially by two enzymatic activities contained within one bifunctional enzyme called PAM (peptidylglycine alpha-amidating mono-oxygenase). Drosophila head extracts contained both of these PAM-related enzyme activities: a mono-oxygenase (PHM) and a lyase (PAL). However, no bifunctional PAM protein was detected. We identified cDNAs encoding an active mono-oxygenase that is highly homologous to mammalian PHM. PHM-like immunoreactivity was found within diverse larval tissues, including the CNS, endocrine glands, and gut epithelium. Northern and Western blot analyses demonstrate RNA and protein species corresponding to the cloned PHM, but not to a bifunctional PAM, leading us to predict the existence of separate PHM and PAL genes in Drosophila. The Drosophila PHM gene displays an organization of exons that is highly similar to the PHM-encoding portion of the rat PAM gene. Genetic analysis was consistent with the prediction of separate PHM and PAL gene functions in Drosophila: a P element insertion line containing a transposon within the PHM transcription unit displayed strikingly lower PHM enzyme levels, whereas PAL levels were increased slightly. The lethal phenotype displayed by the dPHM P element insertion indicates a widespread essential function. Reversion analysis indicated that the lethality associated with the insertion chromosome likely is attributable to the P element insertion. These combined data indicate a fundamental evolutionary divergence in the genes coding for critical neurotransmitter biosynthetic enzymes: in Drosophila, the two enzyme activities of PAM are encoded by separate genes.

Amides↗
PubMed1997

Expression of RESP18 in peptidergic and catecholaminergic neurons.

We examined the expression of regulated endocrine-specific protein of 18-kD (RESP18) in selected peptidergic and catecholaminergic neurons of adult rat brain. In the hypothalamic paraventricular, supraoptic, and accessory nuclei, RESP18 mRNA was highly expressed in neurons immunostained for oxytocin and vasopressin. RESP18 mRNA was also highly expressed in paraventricular nucleus neurons immunostained for corticotropin-releasing hormone, thyrotropin-releasing hormone, and somatostatin. RESP18 mRNA was expressed in POMC cells of the arcuate nucleus, in neuropeptide Y cells of the dorsal tegmental nucleus, lateral reticular nucleus, and hippocampus, and in brainstem catecholaminergic neurons. RESP18 mRNA expression was high in all paraventricular and arcuate neurons, but RESP18 protein was detectable in the perikarya of a subset of these neurons, suggesting an important post-transcriptional component to the regulation of RESP18 expression. RESP18 antisera immunostained perikarya but not axon fibers or terminals. Sub-cellular fractionation of homogenates of several hypothalamic nuclei identified RESP18 protein in fractions enriched in endoplasmic reticulum. The presence of 22- and 24-kD RESP18 isoforms in the neural lobe of the pituitary indicated that some RESP18 protein exited the endoplasmic reticulum. The post-transcriptional regulation of RESP18 expression and localization of RESP18 protein primarily to the endoplasmic reticulum suggests that RESP18 plays a regulatory role in peptidergic neurons.

Adrenergic Fibers↗
PubMed1997

A novel neuroendocrine intracellular signaling pathway.

Expression of many components of the secretory pathway in peptidergic neuroendocrine cells is precisely controlled in response to secretagogues. Regulated endocrine-specific protein (RESP18) was identified as a dopamine-regulated intermediate pituitary transcript. Although the amino acid sequence of RESP18 initially suggested that it might be a novel preprohormone, its widespread expression in peptide-producing neurons and endocrine cells and its localization to the lumen of the endoplasmic reticulum suggested that it subserves a unique function. Subtractive hybridization of a pituitary corticotrope AtT-20 cell line engineered for inducible RESP18 expression demonstrated a RESP18-dependent induction of several transcripts. Regulation of RESP18 expression in vitro and in vivo was accompanied by changes in the same transcripts. Several cDNAs encoding transcripts up-regulated by RESP18 were analyzed by DNA sequencing, searching the GenBank databases for homologous proteins, and Northern blotting. One novel clone showed a tissue distribution nearly identical to that of RESP18. One clone was identical to rat LIMK2, a protein kinase containing modular protein-protein interaction LIM (lin-11, isl-1, mec-3) domains. Another clone was similar to monomeric bacterial isocitrate dehydrogenases. Like the unfolded protein response, these data demonstrate a novel signaling pathway from the secretory pathway lumen to the nucleus. RESP18 acts as a lumicrine peptide (an intracellular luminal autocrine hormone) inducing this pathway.

Amino Acid Sequence↗
PubMed1997

Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

Adrenal Cortex↗
PubMed1997

The expression of regulated endocrine-specific protein of 18 kDa in peptidergic cells of rat peripheral endocrine tissues and in blood.

We examined the cellular localization of regulated endocrine-specific protein of 18 kDa (RESP18) and mRNA in peripheral endocrine tissues. In situ hybridization and immunocytochemistry identified RESP18 mRNA in most cells of the anterior and intermediate pituitary, with RESP18 protein apparent in many anterior pituitary cells but very few intermediate pituitary cells. In the adrenal medulla and superior cervical ganglion, RESP18 mRNA co-localized with dopamine beta-mono-oxygenase and neuropeptide Y. In the thyroid, RESP18 mRNA was localized to C-cells. RESP18 mRNA was expressed in most of the cells of the pancreatic islets, co-localizing with insulin, glucagon, and somatostatin. No RESP18 mRNA or protein was detected in the adrenal cortex, ovary, neural lobe of the pituitary, parathyroid, exocrine pancreas, thyroid follicular cells, placenta, mammary tissue, liver, lung, or atria. As in the intermediate lobe of the pituitary, high levels of RESP18 mRNA in the pancreatic islets and adrenal medulla did not always correlate with immunodetectable RESP protein, suggesting that post-transcriptional mechanisms are important in controlling RESP18 expression. Western blot analyses identified 18 kDa RESP and higher molecular weight isoforms of RESP in most tissues and in plasma. Subcellular fractionation of the anterior pituitary identified 18 kDa RESP18 in fractions enriched in endoplasmic reticulum and secretory granules, with the higher molecular weight isoforms of RESP18 concentrated in fractions enriched in secretory granules. The broad neuroendocrine distribution of RESP18 suggests that it subserves an important function in the secretory pathway that is common to the production of many secreted peptides.

Adrenal Glands↗
PubMed1996

Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme.

The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

Amino Acid Sequence↗
PubMed1996

Identification of routing determinants in the cytosolic domain of a secretory granule-associated integral membrane protein.

We have investigated the trafficking of integral membrane peptidylglycine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal alpha-amidation of peptidylglycine substrates yielding amidated products stored in secretory granules. As soluble proteins, both catalytic domains were independently targeted to secretory granules. In contrast, membrane PAM was largely localized to the trans-Golgi network (TGN). Upon truncation of its cytoplasmic COOH-terminal domain, membrane PAM was less efficiently cleaved by secretory granule enzymes and accumulated on the plasma membrane. When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac protein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane. To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM proteins containing truncations, deletions, or point mutations in the COOH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins accumulated on the plasma membrane, were not efficiently internalized, and were cleaved to generate a bifunctional PAM protein that was not stored in secretory granules. A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-length integral membrane PAM by secretory granule enzymes. Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished cleavage of membrane PAM by secretory granule enzymes and eliminated internalization of PAM from the plasma membrane. The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving membrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for targeting the protein to secretory granules.

Amino Acid Sequence↗
PubMed1996

Prevalence and turnover of peptidylglycine alpha-amidating monooxygenase mRNA in atrial cardiomyocytes.

Peptidylglycine alpha-amidating monooxygenase (PAM), the enzyme responsible for the alpha-amidation of neuroendocrine peptides, is more prevalent in the atrium of the heart than in pituitary or brain. RNase protection assays indicate that PAM transcripts account for approximately 0.5% of the mRNA in the neonatal atrium and 0.06% of the mRNA in the neonatal ventricle. In primary atrial cardiomyocyte cultures PAM mRNA turns over slowly, with a half-life of approximately 20 h. Levels of PAM mRNA in primary atrial cardiomyocytes are increased to 16.5% of control upon treatment with dexamethasone and decreased to 63% of control upon treatment with thyroid hormone.

Animals↗
PubMed1996

Location of neurons that express regulated endocrine-specific protein-18 in the rat diencephalon.

In situ hybridization for regulated endocrine-specific protein-18 messenger RNA showed a distinct and limited pattern of expression in the hypothalamus, midline thalamus, amygdala and hippocampus of the rat. High levels of regulated endocrine-specific protein-18 messenger RNA were found in the magnocellular neurons of the hypothalamic paraventricular, supraoptic and accessory nuclei, in the neurons of the periventricular, medial tuberal, arcuate, lateral and perifornical nuclei, infundibular stalk, and in the ventrolateral division of the ventromedial nucleus and compact division of the dorsomedial nucleus. Lower levels of regulated endocrine-specific protein-18 messenger RNA were found in the parvocellular divisions of the paraventricular nucleus as well as in the bed nucleus of the stria terminalis, median preoptic nucleus, medial preoptic nucleus, medial and lateral preoptic areas, subfornical organ, suprachiasmatic nucleus, anterior hypothalamic area, zona incerta, ventromedial nucleus, dorsomedial nucleus and tuber cinereum. Regulated endocrine-specific protein-18 messenger RNA was also found in thalamic structures including the paraventricular, central medial, intermediodorsal, anterodorsal, rhomboid and reticular nuclei. Signal was also identified in the medial and lateral habenula, in the central, medial, basomedial and anterior cortical nuclei of the amygdala, and in the CA1-CA3 and dentate gyrus of the hippocampus. Dopamine may regulate regulated endocrine-specific protein-18 expression in the CNS because (i) regulated endocrine-specific protein-18 was originally identified in melanotropes based on its regulation by dopaminergic agents and (ii) many of the nuclei that contain regulated endocrine-specific protein-18 also receive dopaminergic input. The localization of regulated endocrine-specific protein-18 in the diencephalon suggests that regulated endocrine-specific protein-18 is involved in regulation of limbic and autonomic function, neuroendocrine control of salt and water balance, reproductive function and feeding behavior.

Amygdala↗
PubMed1996

Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary.

The biosynthesis of peptides requires the synthesis of the prohormone, several biosynthetic processing enzymes, and other granule constituents. We have investigated the regulated expression of proopiomelanocortin (POMC) and five enzymes essential for the processing of POMC to smaller, bioactive peptides in intermediate pituitary melanotropes. Rats were treated with a dopaminergic agonist (bromocriptine) or antagonist (haloperidol) for periods ranging from 1 h to 5 days, followed by analyses of mRNA levels and protein biosynthetic rates. Multiplex RNase protection assays showed that bromocriptine treatment caused a striking decrease in POMC mRNA levels, and significant decreases in mRNA levels for prohormone convertase 2 (PC2), carboxypeptidase H (CPH), and peptidylglycine alpha-amidating monooxygenase (PAM). Smaller increases in mRNA levels were seen after haloperidol stimulation. Protein biosynthetic rates changed more profoundly than mRNA levels at short drug treatment times, indicating a role for translational effects after treatment with bromocriptine and with haloperidol. The homogeneous population of melanotropes in the intermediate lobe of the pituitary allows a quantitative analysis of transcript levels and biosynthetic rates. POMC mRNA levels are 200-1,000-fold higher than levels of any of the processing enzyme mRNAs, and POMC biosynthetic rates exceed those of PC2, PC1, and PAM by 1,000-10,000-fold.

Animals↗
PubMed1996

Identification of the promoter for the gene encoding the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase.

The gene encoding rat peptidylglycine alpha-amidating monooxygenase (PAM) contains 26 protein-coding exons. We identified two non-overlapping genomic clones encoding the 5' untranslated region (UTR) of the PAM gene. Exon 1 has 69 nucleotides flanked by perfect splice acceptor and donor sites, with a TATA motif 25 nucleotides upstream. Exon 0 lacks TATA or CAAT motifs and is embedded in a G + C-rich 800-nucleotide CpG island. The major products identified by RNase protection initiated in exon 0; only a minority of mRNAs initiated in exon 1. 5'-rapid amplification of cDNA ends (RACE) identified the same major transcriptional start sites in exon 0 in the atrium and neurointermediate pituitary. The 2.0-kb fragment upstream of exon 0 and the 1.3-kb fragment upstream of exon 1 were placed upstream of a luciferase-based reporter gene in both sense and antisense orientations. Expression of luciferase was observed in neuroendocrine and nonneuroendocrine cells with both sense constructs. A 0.2-kb fragment of the exon 0 PAM promoter containing multiple GC box elements supported expression of luciferase activity in all cell types. Expression of reporter genes in cells that do not normally express PAM suggests a need for more upstream or intronic information, a role for methylation, or a need for chromatin scaffolding for tissue-specific expression of the endogenous gene.

Animals↗
PubMed1995

Phosphorylation of the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the COOH-terminal alpha-amidation of neural and endocrine peptides through a two-step reaction carried out sequentially by its monooxygenase and lyase domains. PAM occurs in soluble and integral membrane forms. Metabolic labeling of stably transfected hEK-293 and AtT-20 cells showed that [32P]PO4(3-) was efficiently incorporated into Ser and Thr residues of membrane PAM but not into soluble PAM. Truncation of integral membrane PAM proteins (which terminate with Ser976) at Tyr936 eliminated their phosphorylation, suggesting that the COOH-terminal region of the protein was the site of phosphorylation. Recombinant PAM COOH-terminal domain was phosphorylated on Ser932 and Ser937 by protein kinase C (PKC). PAM-1 protein recovered from different subcellular fractions of stably transfected AtT-20 cells was differentially susceptible to calcium-dependent, staurosporine-inhibitable phosphorylation catalyzed by endogenous cytosolic protein kinase(s). Although phorbol ester treatment of hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a bifunctional 105-kDa PAM protein, the effect was an indirect one since it was also observed in hEK-293 cells expressing a truncated PAM-1 protein that was not phosphorylated. AtT-20 cells expressing PAM-1 lacking one of the PKC sites (PAM-1/Ser937-->Ala) exhibited an altered pattern of PAM.PAM antibody internalization, with the mutant protein targeted to lysosomes upon internalization. Thus, phosphorylation of Ser937 in the COOH-terminal cytosolic domain of membrane PAM plays a role in a specific step in the targeting of this protein.

Alanine↗
PubMed1995

A neuroendocrine-specific protein localized to the endoplasmic reticulum by distal degradation.

Regulated endocrine-specific protein, 18-kDa (RESP18), was previously cloned from rat neurointermediate pituitary based on its coordinate regulation with proopiomelanocortin and neuroendocrine specificity. RESP18 has no homology to any known protein. Although RESP18 is translocated across microsomal membranes after in vitro translation, AtT-20 pituitary tumor cells, which endogenously synthesize RESP18, do not release it into the culture medium. In this work, immunostaining and subcellular fractionation have identified RESP18 as an endoplasmic reticulum (ER) protein. Biosynthetic labeling and temperature block studies of AtT-20 cells demonstrated the localization of RESP18 to the ER lumen by a unique mechanism, degradation by proteolysis in a post-ER pre-Golgi compartment. Proteases in this compartment were saturated by exogenous RESP18 overexpression in AtT-20 cells. Furthermore, a calpain protease inhibitor enhanced secretion of RESP18 from AtT-20 cells overexpressing RESP18. Saturation and inhibition of the RESP18 degrading proteases allowed RESP18 to enter secretory granules and acquire a post-translational modification, likely O-glycosylation; this modified 21-kDa RESP18 isoform was the only RESP18 secreted. Rat anterior pituitary extracts contain 18-kDa and O-glycosylated RESP18 with similar properties. Exogenous RESP18 expression in hEK-293 cells demonstrated ER localization and RESP18 metabolism similar to AtT-20 cells, indicating that the cellular machinery involved in localizing RESP18 is not specific to neuroendocrine cells. The data implicate a novel ER localization mechanism for this neuroendocrine-specific luminal ER resident.

Animals↗
PubMed1995

Addition of an endoplasmic reticulum retention/retrieval signal does not block maturation of enzymatically active peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neural and endocrine peptides via a two-step reaction carried out in sequence by the monooxygenase and lyase domains contained in this bifunctional protein. Peptide alpha-amidation is thought to take place primarily in the secretory granules in which mature bioactive peptides are stored, and it is not known where in the secretory compartment newly synthesized PAM protein becomes enzymatically active. To address this question, PAM-3, a soluble bifunctional protein, was modified by addition of the KDEL endoplasmic reticulum (ER) retention/retrieval signal to its COOH terminus. PAM-3-KDEL protein stably expressed in hEK-293 cells or in AtT-20 cells was efficiently retained in the ER based on immunocytochemistry, pulse-chase experiments, and maintained endoglycosidase H sensitivity. The effect of the KDEL sequence was specific since PAM-3 with an inactive ER retention/retrieval signal (PAM-3-KDEV) moved through the secretory pathway like wild type PAM-3. In AtT-20 cells, PAM-3-KDEL was not subjected to the COOH-terminal endoproteolytic cleavage that generates a 75-kDa PAM protein from PAM-3 and PAM-3-KDEV. PAM-3-KDEL protein exhibited both monooxygenase and lyase activities with specific activities similar to those of the wild type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM catalyzes a reaction that occurs primarily in the secretory granules, newly synthesized PAM protein becomes enzymatically competent in the ER.

Amino Acid Sequence↗
PubMed1995

The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of PHM was found not to extend beyond Asp359. Shorter PHM proteins were eliminated intracellularly, suggesting that they failed to fold correctly. A set of mutant PHM proteins whose design was based on the structural and mechanistic similarities of PHM and dopamine beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/D beta M homology domain eliminated activity, while deletion of a third His-rich cluster unique to PHM failed to affect activity; the catalytically inactive mutant PHM proteins still bound to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized PHM indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of PHM and D beta M. Mutation of the Met residues common to PHM and D beta M to Ile identified Met314 as critical for catalytic activity.

Amino Acid Sequence↗
PubMed1995

The NH2-terminal proregion of peptidylglycine alpha-amidating monooxygenase facilitates the secretion of soluble proteins.

A highly conserved ten amino acid proregion separates the peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain of the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein from the NH2-terminal signal peptide; propeptides with amino acid sequences similar to the PAM proregion have been identified in other secreted proteins. In AtT-20 cells, but not in human embryonic kidney (hEK)-293 cells, an endogenous endoprotease acting at a site distal to the trans-Golgi network efficiently removes the propeptide from stably transfected monofunctional PHM (PHMs). We constructed a mutant PHM protein (delta ProPHMs) in which the proregion was deleted and the signal peptide joined directly to the monooxygenase domain. Newly synthesized, enzymatically active delta ProPHMs was secreted from both AtT-20 cells and hEK-293 cells more slowly than PHMs. In endocrine cells, the proregion was not required for storage in regulated secretory granules. We transferred the PAM proregion to prohormone convertase 2 (PC2), another soluble constituent of secretory granules, to determine whether the effect of the proregion were transferrable. In both AtT-20 cells and hEK-293 cells, the PAM/PC2 fusion molecule was able to exit the endoplasmic reticulum more rapidly than PC2.

Amino Acid Sequence↗
PubMed1995

Identification of subcellular compartments containing peptidylglycine alpha-amidating monooxygenase in rat anterior pituitary.

Both soluble and integral membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) are expressed in the rat anterior pituitary, making it an ideal model system for studying the routing of proteins into secretory granules. To identify the subcellular compartments involved in the routing of integral membrane PAM, we used subcellular fractionation, metabolic labeling and immunoblot analysis. Mature secretory granules were found to contain full-length integral membrane PAM along with a significant amount of soluble PAM generated by endoproteolytic cleavage. PAM proteins were not co-distributed with tyrosylprotein sulfotransferase activity during sucrose gradient centrifugation, indicating that the trans-Golgi/TGN is not a major PAM-containing compartment at steady state. Fractionation of the 4,000 g and 10,000 g pellets obtained by differential centrifugation identified a significant amount of integral membrane PAM in a light fraction lacking soluble secretory granule proteins. Metabolic labeling experiments with primary anterior pituitary cells demonstrated that integral membrane PAM enters a light compartment with similar properties only after exit from the trans-Golgi/TGN. Comparison of the metabolic labeling and immunoblot analyses suggests that PAM in this post-trans-Golgi/TGN compartment is in organelles involved in the intracellular recycling of integral membrane PAM. Small amounts of full-length integral membrane PAM were also recovered in fractions containing internalized transferrin and may be in an endosomal compartment following retrieval from the cell surface.

Animals↗