Carboxypeptidase E is a sorting receptor for prohormones: binding and kinetic studies

Area of Science:

• Endocrinology
• Molecular Biology
• Cell Biology

Background:

• The regulated secretory pathway (RSP) is crucial for hormone and neuropeptide release.
• Carboxypeptidase E (CPE) is a key enzyme implicated in the sorting of cargo to secretory granules.
• Understanding CPE's interactions is vital for elucidating neuroendocrine secretion mechanisms.

Purpose of the Study:

• To investigate the binding characteristics of CPE to various prohormones within pituitary secretory granule (SG) membranes.
• To determine if CPE acts as a common sorting receptor for prohormones destined for the RSP.

Main Methods:

• Investigated the binding of pro-opiomelanocortin (POMC), pro-insulin, pro-enkephalin, and chromogranin A (CGA) to bovine pituitary SG membrane CPE.
• Utilized binding assays to assess affinity and kinetics, including ion dependency.
• Employed crosslinking studies to confirm specific interactions.
• Used extraction methods (NaHCO3, KSCN, Triton X-100/NaCl) to assess CPE's association with SG membranes.

Main Results:

• N-POMC1-26, pro-insulin, and pro-enkephalin bound to membrane CPE with similar low affinity and ion-independent kinetics.
• Pro-enkephalin demonstrated similar IC50 values to pro-insulin and N-POMC1-26, while CGA did not bind.
• Crosslinking confirmed specific binding of pro-insulin and pro-enkephalin to SG membrane CPE.
• CPE was tightly associated with SG membranes, extractable with NaHCO3 or KSCN.

Conclusions:

• Membrane-bound CPE exhibits characteristics of a common sorting receptor for multiple prohormones.
• CPE's ability to bind various prohormones, excluding CGA, supports its role in targeting cargo to the regulated secretory pathway.
• These findings provide further evidence for CPE's function in prohormone sorting and neuroendocrine secretion.