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

Abstract

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 two-fold 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. Co-immunoprecipitation 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.

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

“…4). As in our previous study (7), the ␥ 3 MSH antibody detected similar amounts of differentially glycosylated POMC and 16-kDa fragment in lysates of Scr and sh-1A PAM-1 cells; consistent with previous metabolic labeling experiments, ABI and 18-kDa fragment were barely detectable in cell lysates (Fig. 4A).…”

“…3A). The TGN region of sh-1A PAM-1 cells is vacuolated; noncondensing secretory granules accumulate at the expense of immature secretory granules, and secretagogue-stimulated secretion is diminished (7). Although decreased expression of 1A resulted in lower levels of another AP-1 subunit (␥-adaptin), we confirmed that levels of ␣-adaptin, an AP-2 subunit, were unaffected (Fig.…”

“…Generation of sh-1A and scramble PAM-1 AtT-20 cells using lentivirus targeted to 1A (Sigma; clone ID TRCN0000111549) and a nontarget shRNA (Sigma, catalog no. SHC002V), respectively, was described previously (7).…”

“…In both cell lines, the amidation of 18-kDa fragment by PAM must occur in the brief time following its cleavage from ABI and before its cleavage into 16-kDa fragment and JP-Gly. With normal levels of AP-1, this step occurs as 18-kDa fragment traverses immature secretory granules, a major site linking the biosynthetic and endocytic pathways (7).…”

“…Using shRNA to decrease expression of the 1A subunit to half, AP-1 was shown to play an essential role in secretory granule biogenesis and PAM trafficking in AtT-20 mouse corticotrope tumor cells (6,7,14). Although ATP7A trafficking in neuroendocrine cells has not been extensively explored, studies in other cell types predict an essential role for AP-1 (6,14,16,17).…”