Identification of Routing Determinants in the Cytosolic Domain of a Secretory Granule-associated Integral Membrane Protein

Abstract

We have investigated the trafficking of integral membrane peptidylglycine ␣-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal ␣-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 ␣-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.

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.…”

Kalirin, a Multifunctional PAM COOH-terminal Domain Interactor Protein, Affects Cytoskeletal Organization and ACTH Secretion from AtT-20 Cells

“…The rabbit polyclonal antibody was visualized with FITC-tagged goat anti-rabbit F(abЈ) 2 IgG(HϩL) (Caltag Laboratories, Burlingame, CA), and the monoclonal antibody was visualized with Cy TM 3-tagged AffiniPure donkey anti-mouse IgG(HϩL) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) (41). Antibody uptake experiments with AtT-20 cells have been described previously (9). Actin filaments were visualized in AtT-20 and CHO cells fixed in 3.7% paraformaldehyde for 10 to 30 min using FITC-/halloidin (0.125 to 0.5 g/ml) (13,27,42,43 …”

“…Duplicate wells of cells were plated on polylysine-coated wells for release experiments. To study secretion of ACTH, wells were incubated for three 30-min periods in basal release medium (Dulbecco's modified Eagle's medium/F12-Air with 2 mg/ml fatty acid-free bovine serum albumin, 0.1 mg/ml lima bean trypsin inhibitor, 1 g/ml insulin, 0.1 g/ml transferrin), and the medium was discarded (8,37,38). Then 30-min collections were performed in control medium, in medium containing 100 nM corticotropinreleasing hormone (Sigma), or 1 mM BaCl 2 .…”

“…Immunocytochemical Procedures-AtT-20 cells plated onto polylysine-coated chamber slides were fixed with ice-cold methanol or warm 4% paraformaldehyde and stained as described previously (9,41). Dual staining used a rabbit polyclonal antiserum for Kalirin (Ab2581, 1:1000) and a mouse monoclonal antibody for ␥-adaptin (Transduction Laboratories, Lexington, KY).…”

“…main of PAM immediately following its single transmembrane domain yields active bifunctional enzyme that accumulates on the plasma membrane, unable to adopt its normal transGolgi network and secretory granule localization or to undergo endocytosis (9).…”