Identification of the Sorting Signal Motif within Pro-opiomelanocortin for the Regulated Secretory Pathway (*)

The NH2-terminal region of pro-opiomelanocortin (POMC) is highly conserved across species, having two disulfide bridges that cause the formation of an amphipathic hairpin loop structure between the 2nd and 3rd cysteine residues (Cys8to Cys). The role that the NH2-terminal region of pro-opiomelanocortin plays in acting as a molecular sorting signal for the regulated secretory pathway was investigated by using site-directed mutagenesis either to disrupt one or more of the disulfide bridges or to delete the amphipathic loop entirely. When POMC was expressed in Neuro-2a cells, ACTH immunoreactive material was localized in punctate secretory granules in the cell body and along the neurites, with heavy labeling at the tips. ACTH was secreted from these POMC-transfected cells in a regulated manner. Disruption of both disulfide bridges or the second disulfide bridge or removal of the amphipathic hairpin loop resulted in constitutive secretion of the mutant POMC from the cells and a lack of punctate secretory granule immunostaining within the cells. We have modeled the NH2-terminal POMC Cys8to Cysdomain and have identified it as an amphipathic loop containing four highly conserved hydrophobic and acidic amino acid residues (Asp-Leu-Glu14-Leu18). Thus the sorting signal for POMC to the regulated secretory pathway appears to be encoded by a specific conformational motif comprised of a 13-amino acid amphipathic loop structure stabilized by a disulfide bridge, located at the NH2terminus of the molecule.

[1]  D. Cool,et al.  Identification of a sorting signal for the regulated secretory pathway at the N-terminus of pro-opiomelanocortin , 1994, Biochimie.

[2]  W. Huttner,et al.  The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans‐Golgi network , 1994, FEBS letters.

[3]  Loh Yp,et al.  The amino-terminal sequence of pro-opiomelanocortin directs intracellular targeting to the regulated secretory pathway. , 1993 .

[4]  P. Crine,et al.  Targeting of pro-opiomelanocortin to the regulated secretory pathway may involve cooperation between different protein domains , 1993, Molecular and Cellular Endocrinology.

[5]  W. Huttner,et al.  Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans‐Golgi network causes its missorting to the constitutive secretory pathways. , 1993, The EMBO journal.

[6]  H. Akil,et al.  Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA , 1993, Neuropeptides.

[7]  R. Kelly.  Storage and release of neurotransmitters , 1993, Cell.

[8]  M. Yilla,et al.  Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system , 1992, The Journal of cell biology.

[9]  A. Helenius,et al.  Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum , 1992, The Journal of cell biology.

[10]  Y. Loh,et al.  Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme. , 1992, The Journal of biological chemistry.

[11]  W. Huttner,et al.  Chromogranin B (secretogranin I), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in PC12 cells. , 1992, The Journal of biological chemistry.

[12]  P. Crine,et al.  Investigation of a possible role of the amino-terminal pro-region of proopiomelanocortin in its processing and targeting to secretory granules , 1991, Molecular and Cellular Endocrinology.

[13]  P. Arvan,et al.  Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. , 1991, The Journal of biological chemistry.

[14]  P. Crine,et al.  Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: Targeting of the foreign neuropeptide to dense-core vesicles , 1991, Molecular and Cellular Endocrinology.

[15]  W. Sossin,et al.  Sorting within the regulated secretory pathway occurs in the trans- Golgi network , 1990, The Journal of cell biology.

[16]  R. Mains,et al.  Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells. , 1989, Molecular endocrinology.

[17]  D. Shields,et al.  The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells , 1989, The Journal of cell biology.

[18]  E. Rassart,et al.  Targeting and Processing of Pro‐Opiomelanocortin in Neuronal Cell Lines , 1989, Journal of neurochemistry.

[19]  S. Wray,et al.  Prenatal ontogenesis of pro-opiomelanocortin in the mouse central nervous system and pituitary gland: an in situ hybridization and immunocytochemical study. , 1989, Brain research. Developmental brain research.

[20]  P. Walter,et al.  Molecular sorting in the secretory pathway. , 1989, Science.

[21]  G. Soma,et al.  Absence of a γ-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta , 1988 .

[22]  D. L. Quinn,et al.  The trans-most cisternae of the Golgi complex: A compartment for sorting of secretory and plasma membrane proteins , 1987, Cell.

[23]  R. Kelly,et al.  In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules , 1987, The Journal of cell biology.

[24]  N. Seidah,et al.  Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1-76). , 2009, International journal of peptide and protein research.

[25]  R. Kelly.  Pathways of protein secretion in eukaryotes. , 1985, Science.

[26]  Y. Loh,et al.  Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. , 1985, The Journal of biological chemistry.

[27]  J. Douglass,et al.  Polyprotein gene expression: generation of diversity of neuroendocrine peptides. , 1984, Annual review of biochemistry.

[28]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[29]  H. Kawauchi.  Chemistry of proopiocortin-related peptides in the salmon pituitary. , 1983, Archives of biochemistry and biophysics.

[30]  H. Gainer,et al.  Synthesis, transport, and release of posterior pituitary hormones. , 1980, Science.

[31]  F. Ruddle,et al.  Isolation of microtubule protein from cultured mouse neuroblastoma cells. , 1970, Proceedings of the National Academy of Sciences of the United States of America.