Secretion Stimulates Intramembrane Proteolysis of a Secretory Granule Membrane Enzyme

Abstract

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 ␣-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a ␥-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 NH 2 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 ␣-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a ␥-secretase-like cleavage. Cleavage by ␥-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.Cells utilize diverse signaling mechanisms to coordinate the functions of their subcellular organelles and to integrate their metabolism with that of neighboring cells and distant tissues. Regulated intramembrane proteolysis, which is performed by a small group of intramembrane cleaving proteases (i-CLiPs), 2 initiates many of the signaling cascades involved in these pathways. The i-CLiP family includes metalloproteases (S2P), rhomboid serine proteases, signal peptide peptidase aspartyl proteases, and ␥-secretase aspartyl protease complexes (1, 2). For example, signaling pathways that regulate lipid homeostasis modulate cleavage of SREBP by S2P, releasing its transcription factor domain. The unfolded protein response involves S2P-mediated cleavage of ATF6 and nuclear translocation of its N-terminal transcription factor region (3). Many aspects of development involve cleavage of Notch by ␥-secretase, generating a cytosolic domain fragment that translocates to the nucleus, where it participates in the regulation of gene transcription (4). The ␥-secretase-mediated cleavage of amyloid precursor protein (APP) generates amyloidogenic fragments along with a cytosolic fragment whose role is under investigation (5).Secretory granules in the regulated exocytic pathway play an essential role in maintaining homeostasis. Their soluble content includes chromogranins, prohormone conve...

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

“…AtT-20 cells must deliver copper to the secretory pathway to support the synthesis of amidated peptides. Immunoelectron microscopy previously established the presence of PAM-1 in the tubulovesicular structures that constitute the TGN cisternae, immature secretory granules, and multivesicular bodies (24,31,32). Atp7a also concentrated in the perinuclear region, where its distribution overlapped that of the cis-Golgi marker, GM130, and ␥-adaptin ( Fig.…”

“…The Golgi region of AtT-20 cells includes immature secretory granules as well as a complex mixture of endocytic compartments (31,32). Methods for quantifying the trafficking of PAM⅐antibody complexes formed on the cell surface through early endosomes and into multivesicular bodies were developed previously, revealing regulated movement of these complexes from the external membrane of the multivesicular body into intraluminal vesicles (31).…”

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking

“…Cells were incubated with MG132 (2 M; added from a 10 mg/ml stock in DMSO; Sigma) for 4.5 h. Cells were then extracted into ice-cold TMT containing protease inhibitors (33); lysates were allowed to tumble for 30 min at 4°C and then clarified. Cells were also incubated with NH 4 Cl (20 mM; added from a 160 mM isotonic stock in water), concanamycin A (1 nM, added from a 10 M stock in DMSO; Sigma), lactacystin (10 M, added from a 10 mM stock in water; Sigma), or leupeptin (20 M, added from a 2 mM stock in water; Sigma).…”

“…Antigen-antibody complexes were detected using horseradish peroxidase-conjugated secondary antibody and Super Signal West Pico chemiluminescent substrate (Pierce). Affinity-purified rabbit poly-clonal antibodies (1:1000 dilution for each) used to visualize PAM included the following: PHM antibody JH1761 (raised to rPAM-1(37-382)) (5); exon 16 antibody JH629 (raised to rPAM-1(394 -498)) (3); PAL antibody JH471 (raised to rPAM-1(463-864)) (5); and CD antibody CT267 (raised to rPAM-1(965-976)) (33).…”

“…Membranes were prepared by differential centrifugation (33). Pellets were suspended in 20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 0.02% Nonidet P-40.…”