Differential trafficking of soluble and integral membrane secretory granule-associated proteins

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.     

Targeting of membrane proteins to the regulated secretory pathway in anterior pituitary endocrine cells

Unlike the neuroendocrine cell lines widely used to study trafficking of soluble and membrane proteins to secretory granules, the endocrine cells of the anterior pituitary are highly specialized for the production of mature secretory granules. Therefore, we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the regulated secretory pathway even when expressed at levels 40-fold higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking the lumenal, catalytic domains of PAM, is still stored in granules. Secretory granules are not the default pathway for all membrane proteins, because Tac accumulates on the surface of pituitary endocrine cells. Overexpression of PAM is accompanied by a diminution in its endoproteolytic cleavage and in its BaCl(2)-stimulated release from mature granules. Because internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or Myc-TMD/CD alters the organization of the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.     

Secretion Stimulates Intramembrane Proteolysis of a Secretory Granule Membrane Enzyme

Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH₂ termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.     

Induction of Integral Membrane PAM Expression in AtT-20 Cells Alters the Storage and Trafficking of POMC and PC1

Peptidylglycine α-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH₂-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.     

Expression of a peptide processing enzyme in cultured cells: truncation mutants reveal a routing domain.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme responsible for the alpha-amidation of peptides in secretory granules of neuroendocrine cells. The single gene encoding PAM undergoes tissue-specific alternative splicing and endoproteolytic processing to generate bifunctional membrane proteins with a single transmembrane domain as well as soluble proteins that are mono- or bifunctional. In order to examine the endoproteolytic processing and subcellular localization of the various forms of PAM in cells lacking regulated secretory granules, we established stably transfected hEK-293 cell lines expressing naturally occurring and mutant forms of PAM. As expected, newly synthesized soluble PAM proteins were rapidly secreted into the medium. Integral membrane protein forms of PAM were largely localized in the perinuclear region with punctate staining visible throughout the cell and 2-5% of the enzyme activity detectable on the cell surface. Bifunctional PAM proteins were slowly released into the medium after expression of integral membrane protein forms of PAM. Deletion of 77 amino acids from the COOH-terminus of the integral membrane forms of PAM resulted in a membrane-bound protein which retained both enzymatic activities but accumulated on the cell surface. Rapid internalization of full-length PAM proteins was observed by incubating live cells with antiserum to PAM; deletion of the COOH-terminal domain eliminated the ability of cells to internalize PAM. Thus the cytoplasmic domain of integral membrane PAM contains a routing determinant recognized by cells lacking the regulated secretory pathway.     

COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH- terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.     

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.     

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.     

Access of a membrane protein to secretory granules is facilitated by phosphorylation

Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential for the biosynthesis of amidated peptides, was used to assess the role of cytosolic acidic clusters in trafficking to regulated secretory granules. Casein kinase II phosphorylates Ser(949) and Thr(946) of PAM, generating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identified by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific for phospho-Ser(949)-PAM-CD demonstrates that a small fraction of the PAM-1 localized to the perinuclear region bears this modification. Pituitary cell lines expressing PAM-1 mutants that mimic (TS/DD) or prevent (TS/AA) phosphorylation at these sites were studied. PAM-1 TS/AA yields a lumenal monooxygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granules more efficiently than wild-type PAM-1 and monooxygenase release is more responsive to secretagogue. Furthermore, this acidic cluster affects exit of internalized PAM-antibody complexes from late endosomes; internalized PAM-1 TS/DD accumulates in a late endocytic compartment instead of the trans-Golgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking.     

AP‐1A Controls Secretory Granule Biogenesis and Trafficking of Membrane Secretory Granule Proteins

The adaptor protein 1A complex (AP‐1A) transports cargo between the trans‐Golgi network (TGN) and endosomes. In professional secretory cells, AP‐1A also retrieves material from immature secretory granules (SGs). The role of AP‐1A in SG biogenesis was explored using AtT‐20 corticotrope tumor cells expressing reduced levels of the AP‐1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non‐condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue‐stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α‐amidating monooxygenase‐1 (PAM‐1), integral membrane enzymes that enter immature SGs. The non‐condensing SGs contained POMC products and PAM‐1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM‐1 into PHM was unaltered, but PHM basal secretion was increased in sh‐μ1A PAM‐1 cells. Despite lacking a canonical AP‐1A binding motif, yeast two‐hybrid studies demonstrated an interaction between the PAM‐1 cytosolic domain and AP‐1A. Coimmunoprecipitation experiments with PAM‐1 mutants revealed an influence of the luminal domains of PAM‐1 on this interaction. Thus, AP‐1A is crucial for normal SG biogenesis, function and composition.      

Response of an integral granule membrane protein to changes in pH

A key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secretory granules. Because peptidylglycine alpha-amidating monooxygenase (PAM) is one of the few membrane-spanning proteins concentrated in secretory granules and is a known effector of regulated secretion, we examined its sensitivity to pH. Based on antibody binding experiments, the noncatalytic linker regions between the two enzymatic domains of PAM show pH-dependent conformational changes; these changes occur in the presence or absence of a transmembrane domain. Integral membrane PAM-1 solubilized from rat anterior pituitary or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extracts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to low pH-induced aggregation, whereas PAM-1 recovered from a light, intracellular recycling compartment is not. Mutagenesis studies indicate that a transmembrane domain is necessary but not sufficient for low pH-induced aggregation and reveal a short lumenal, juxtamembrane segment that also contributes to pH-dependent aggregation. Taken together, these results demonstrate that several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells.     

Secretory Granule Membrane Protein Recycles through Multivesicular Bodies

The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine α‐amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr⁹⁴⁶, Ser⁹⁴⁹) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100‐kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.     

A Histidine-rich Linker Region in Peptidylglycine α-Amidating Monooxygenase Has the Properties of a pH Sensor

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.     

Sorting ourselves out: seeking consensus on trafficking in the beta-cell

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans -Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.     

Supplying copper to the cuproenzyme peptidylglycine alpha-amidating monooxygenase

We explored the role of known copper transporters and chaperones in delivering copper to peptidylglycine-alpha-hydroxylating monooxygenase (PHM), a copper-dependent enzyme that functions in the secretory pathway lumen. We examined the roles of yeast Ccc2, a P-type ATPase related to human ATP7A (Menkes disease protein) and ATP7B (Wilson disease protein), as well as yeast Atx1, a cytosolic copper chaperone. We expressed soluble PHMcc (catalytic core) in yeast using the yeast pre-pro-alpha-mating factor leader region to target the enzyme to the secretory pathway. Although the yeast genome encodes no PHM-like enzyme, PHMcc expressed in yeast is at least as active as PHMcc produced by mammalian cells. PHMcc partially co-migrated with a Golgi marker during subcellular fractionation and partially co-localized with Ccc2 based on immunofluorescence. To determine whether production of active PHM was dependent on copper trafficking pathways involving the CCC2 or ATX1 genes, we expressed PHMcc in wild-type, ccc2, and atx1 mutant yeast. Although ccc2 and atx1 mutant yeast produce normal levels of PHMcc protein, it lacks catalytic activity. Addition of exogenous copper yields fully active PHMcc. Similarly, production of active PHM in mouse fibroblasts is impaired in the presence of a mutant ATP7A gene. Although delivery of copper to lumenal cuproproteins like PAM involves ATP7A, lumenal chaperones may not be required.     

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H⁺ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.     

Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway.

P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion.     

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.     

Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.