Abstract
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 (Cys8 to Cys20). 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 Cys8 to Cys20 domain and have identified it as an amphipathic loop containing four highly conserved hydrophobic and acidic amino acid residues (Asp10-Leu11-Glu14-Leu1). 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 NH2 terminus of the molecule.
Highlights
Endocrine cells and peptidergic neurons contain a regulated secretory pathway, in addition to the "default" constitutive secretory pathway present in all cells [1]
Expression of POMC in Neuro-2a Cells-When full-length POMC was expressed in Neuro-2a cells [18,19,20,21], ACTH; was immunocytochemically localized to punctate secretory granules in both the main cell body and along the neurites with very heavy punctate labeling at the tips of the neurites, which suggested accumulation in this region (Fig. 2A)
Analysis of the ACTH; products showed processing of POMC to ACTH1- 39 and ACTH1- 14 (Fig. 3). These ACTH products were secreted into the medium in a stimulated manner with high K+/Ca2 + (Table I). These results showed that POMC was sorted to the regulated secretory pathway in Neuro-2a cells, making this a useful cell line in which to study prohormone sorting
Summary
Site-directed Mutagenesis of Bovine POMC-Bovine POMC eDNA Using an Amersham mutagenesis system (version 2, RPN 1523), substitution mutations were made by annealing the ssM13-POMC to a 39-nucleotide probe complementary to positions 72111 in POMC. The probe contained glycine-to-cysteine mutations in position 82, 101, or both, and after annealing was treated with DNA polymerase (K!enow fragment) and T4-ligase to convert the hybrid to dsM13-POMC. Wild type and mutated POMC eDNA were cut from the M13 vector by Psti and blunt ended by T4 polymerase, and Nhei linkers were ligated. The POMC eDNA was ligated to a Nhei- and Smal-digested pMSG expression vector and transfected into HB101 Escherichia coli. Human vasopressin Human oxytocin Rat dynorphin Rat proenkephalin Human chromogranin B wi ·L E s sa LESSa .LESS a LESSa ,LESS a .LESS a LESSa 'WE S S R
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
