A neuroendocrine-specific protein localized to the endoplasmic reticulum by distal degradation.
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.
Schiller
M R
MR
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Mains
R E
RE
Eipper
B A
BA
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D018613:Microscopy, Confocal; D008861:Microsomes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis*; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D010911:Pituitary Neoplasms; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C086381:regulated endocrine secretory protein 18