A Neuroendocrine-specific Protein Localized to the Endoplasmic Reticulum by Distal Degradation

Martin R Schiller,Richard E Mains,Betty A Eipper

doi:10.1074/jbc.270.44.26129

Martin R Schiller, Richard E Mains + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.44.26129

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Abstract

Regulated endocrine-specific protein, 18-kDa (RESP18), was previously cloned from rat neurointermediate pituitary based on its coordinate regulation with proopiomelanocortin and neuroendocrine specificity. RESP18 has no homology to any known protein. Although RESP18 is translocated across microsomal membranes after in vitro translation, AtT-20 pituitary tumor cells, which endogenously synthesize RESP18, do not release it into the culture medium. In this work, immunostaining and subcellular fractionation have identified RESP18 as an endoplasmic reticulum (ER) protein. Biosynthetic labeling and temperature block studies of AtT-20 cells demonstrated the localization of RESP18 to the ER lumen by a unique mechanism, degradation by proteolysis in a post-ER pre-Golgi compartment. Proteases in this compartment were saturated by exogenous RESP18 overexpression in AtT-20 cells. Furthermore, a calpain protease inhibitor enhanced secretion of RESP18 from AtT-20 cells overexpressing RESP18. Saturation and inhibition of the RESP18 degrading proteases allowed RESP18 to enter secretory granules and acquire a post-translational modification, likely O-glycosylation; this modified 21-kDa RESP18 isoform was the only RESP18 secreted. Rat anterior pituitary extracts contain 18-kDa and O-glycosylated RESP18 with similar properties. Exogenous RESP18 expression in hEK-293 cells demonstrated ER localization and RESP18 metabolism similar to AtT-20 cells, indicating that the cellular machinery involved in localizing RESP18 is not specific to neuroendocrine cells. The data implicate a novel ER localization mechanism for this neuroendocrine-specific luminal ER resident.

Highlights

Include lipid synthesis, protein synthesis, protein folding, Ca2ϩ storage, and N-glycosylation [1,2,3,4,5]
RESP18 biosynthesis in dexamethasone-treated AtT-20 corticotrope tumor cells approached the biosynthetic level of the major prohormone precursor, POMC, pulse-chase studies failed to reveal any processing of RESP18 beyond removal of the signal peptide, and no RESP18 or processed products were recovered from spent medium [18]
When anterior pituitary tissue extract was treated with neuraminidase, the 24-kDa RESP18 band was eliminated, and more 18-kDa RESP18 appeared; not reduced to a single band it was clear that glycosylation of RESP18 occurred (Fig. 1C)

Summary

Introduction

Include lipid synthesis, protein synthesis, protein folding, Ca2ϩ storage, and N-glycosylation [1,2,3,4,5]. RESP18 Is an Endoplasmic Reticulum Protein—The localization of RESP18 and BiP/GRP78 was examined in AtT-20 and GH3 cells by dual label confocal microscopy (Fig. 2). RESP18 Is Degraded in Post-ER Pre-Golgi Compartment—As observed previously, RESP18 had a half-life of 18 min in AtT-20 cells incubated at 37 °C [18].

Results

Conclusion