Targeting of Membrane Proteins to the Regulated Secretory Pathway in Anterior Pituitary Endocrine Cells

Abstract

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 ␣-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.Peptidylglycine ␣-amidating monooxygenase (PAM) 1 is a bifunctional enzyme involved in the posttranslational processing of many prohormones and neuropeptides. PAM catalyzes the formation of ␣-amidated peptides from peptide precursor molecules with a COOH-terminal glycine. In neurons and endocrine cells, biologically active peptides are stored in secretory granules that undergo regulated release in response to external stimuli. Localized in secretory granules of many neural and endocrine tissues, PAM is one of a small number of posttranslational processing enzyme occurring naturally in soluble and membrane forms (1-4). For this reason, we have used PAM to investigate the trafficking of soluble and membrane proteins into secretory granules (5, 6).Soluble and membrane PAM are targeted to the regulated secretory pathway in different ways (5, 6). By stably expressing wild type and mutant PAM in AtT-20 corticotrope tumor cells, protein domains containing trafficking information were identified (7-9). The two catalytic domains of PAM can be expressed independently, and both are efficiently targeted to secretory granules. In contrast, membrane PAM is predominantly localized in the trans-Golgi network (TGN) at steady state. The small amount of membrane PAM on the cell surface is rapidly internalized and accumulates in the TGN region (7). The cytosolic domain of PAM contains information necessary for the trafficking of this membrane protein within the secretory pathway (7). Membrane proteins lacking the cytosolic domain are less extensively cleaved by secretory granule endoproteases, accumulate on the plasma membrane, and fail to undergo internalization. A m...

An Arginine Stretch Limits ADAM10 Exit from the Endoplasmic Reticulum

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An NMDA Receptor ER Retention Signal Regulated by Phosphorylation and Alternative Splicing

“…To identify the relevant trafficking signals in NR1 and to minimize effects of NR2 subunits and other NR1 domains, we chose to isolate the contribution of NR1 C-terminal domains using Tac-NR1 chimeras. Such an approach has been used extensively to identify signals involved in secretory and endocytic membrane trafficking (Milgram et al, 1996;Ghosh et al, 1998;Jansen et al, 1998;Tan et al, 1998;El Meskini et al, 2001). The experimental advantages of this ap- proach include the well characterized nature of the Tac protein and its trafficking, the existence of highly specific extracellular epitope antibodies, and the monomeric nature of the Tac chimeras.…”

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