Cytoplasmic Transport Signal is Involved in Phogrin Targeting and Localization to Secretory Granules

Abstract

Phogrin is an integral glycoprotein primarily expressed in neuroendocrine cells. The predominant localization of phogrin is on dense-core secretory granules, and the lumenal domain has been shown to be involved in its efficient sorting to the regulated secretory pathway. Here, we present data showing that a leucine-based sorting signal [EExxxIL] within the cytoplasmic tail contributes its steady-state localization to secretory granules. Deletion mutants in the tail region failed to represent granular distribution in pancreatic b-cell line, MIN6, and anterior pituitary cell line, AtT-20. A sorting signal mutant with two glutamic acids substituted into alanines (EE/AA) is primarily accumulated in the Golgi area instead of secretory granules, and another mutant (IL/AA) is trapped at the plasma membrane due to a defect in endocytosis. We further demonstrate that the leucine-based sorting signal of phogrin specifically interacts with both adaptor protein AP-1 and AP-2 clathrin adaptor complexes in vitro. These observations, along with previous studies, suggest that distinct domains of phogrin mediate proper localization of this transmembrane protein on secretory granules.

Gene Silencing of Phogrin Unveils Its Essential Role in Glucose-Responsive Pancreatic β-Cell Growth

“…Phogrin localizes to insulin-containing secretory granules and translocates to the plasma membrane whenever insulin exocytosis is induced (Fig. 7D) (36). Thus, interaction of phogrin with insulin receptor is coordinately coupled with the autocrine action of insulin.…”

“…Transfections were performed with Lipofectamin 2000 reagent (Invitrogen, Carlsbad, CA). MIN6 cells were transfected with pcDNA3 vector plus pSUPER plasmid, and stable clones were selected in the presence of G418 (36). Isolated colonies of the shPhogrin1-transfected cells were transferred to new culture dishes for propagation (a total of 192 clones: 72 clones in the 1st round, 120 clones in the 2nd round), but only two lines grew up to sufficient scales.…”

“…Immunoprecipitation and immunoblotting analyses. Immunoprecipitation and immunoblot analyses were performed as described previously (36). For immunoprecipitation, MIN6 cells were extracted with lysis buffer (20 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 0.5% Nonidet P-40, 1 mmol/l EGTA, 0.5 mmol/l phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin).…”

Expression and Function of IA-2 Family Proteins, Unique Neuroendocrine-specific Protein-tyrosine Phosphatases

“…Selective packaging of peptide hormones and their associated proteins into SGs may require molecular mechanisms including several sorting domains for specific protein-protein and protein-lipid interactions and selective aggregation of secretory proteins for retention in the granules [37,38]. Recent observations on IA-2β suggest that the luminal pro domain contributes to its sorting into SGs [6] and the trafficking signals within the cytoplasmic tail play an essential role in its steady-state localization to SGs [39,40]. The trafficking signals correspond to a tyrosin-based sorting motif (YQE/DL) and a leucine-based sorting motif (EExxxI/LL), and these sequences are well conserved among the family proteins in the species ( Figure 1) [39,40].…”

“…Recent observations on IA-2β suggest that the luminal pro domain contributes to its sorting into SGs [6] and the trafficking signals within the cytoplasmic tail play an essential role in its steady-state localization to SGs [39,40]. The trafficking signals correspond to a tyrosin-based sorting motif (YQE/DL) and a leucine-based sorting motif (EExxxI/LL), and these sequences are well conserved among the family proteins in the species ( Figure 1) [39,40]. Both cytoplasmic signals are involved in the trafficking of mature IA-2β protein at the plasma membrane (endocytosis), presumably through the interaction with AP-2 clathrin-adaptor complexes [39,40].…”