Carboxypeptidase E, a Prohormone Sorting Receptor, Is Anchored to Secretory Granules via a C-Terminal Transmembrane Insertion

Abstract

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.

A bi-directional carboxypeptidase E-driven transport mechanism controls BDNF vesicle homeostasis in hippocampal neurons

“…The luminal domain of the membrane form of CPE acts as a sorting receptor for targeting BDNF to the regulated secretory pathway vesicles in hippocampal and cortical neurons (Lou et al 2005). Membrane CPE can also exist in vesicles as a transmembrane protein with a cytoplasmic tail of ~10 amino acids at the C-terminus (Arnaoutova et al, 2003;Dhanvantari et al, 2002;Wu et al, 2004). The CPE cytoplasmic tail was found to interact with an activated form of the cytoplasmic small GTPase, Arf6, to recycle CPE and a CPE binding protein, eosinophil cationic protein, from the plasma membrane to the TGN in stimulated neuroendocrine cells (Arnaoutova et al, 2003).…”

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo

“…Numerous integral or membranebound proteins were identified, including several involved in vesicle trafficking and cytoskeletal proteins, as expected. However, potential sorting receptors such as SgIII (Han et al 2008) or carboxypeptidase E (Dhanvantari et al 2002) were not identified in parotid membranes by this method. Nonetheless, one salivary cargo protein, PSP, was identified.…”

“…CPE itself binds the granule membrane. The C-terminus of CPE can span the membrane, although only 5 amino acid residues are cytosolic (Dhanvantari et al 2002). Recycling of CPE from the plasmalemma requires ARF6 apparently due to direct binding to the coat protein.…”

Modulation of Secretory Granule-targeting Efficiency by Cis and Trans Compounding of Sorting Signals

“…These include paired basic residues that may interact with granule-resident PC enzymes (14, 24 -26), N-terminal disulfide-bound loops (27,28), and other domains that mediate sorting by binding to caboxypeptidase E (2), calcium-binding domains that lead to aggregation (reviewed in Ref. 8), and ␣-helices that either traverse or interact with membranes (3,(5)(6)(7). Finally, proteins may contain more than one single type of sorting domain so that analyses in which any one of these is eradicated will not eliminate sorting in the context of the native protein.…”

“…This process, called regulated secretion, depends on a highly selective triage of proteins within or just after the TGN. Retention in dense core secretory granules is not a random process and granulesorted proteins must possess unique properties or must interact with other cellular components in a unique way to end up in this organelle (1)(2)(3)(4)(5)(6)(7).…”