Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts.
Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans -Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Delta4 and CPE-Delta15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Delta4 nor CPE-Delta15 floated in sucrose density gradients. The sorting of both CPE-Delta4 and CPE-Delta15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Delta4 or CPE-Delta15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr(431)-Leu-Asn-Phe(434), are required for raft association and sorting.
Zhang
Chun-Fa
CF
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4480, USA.
Dhanvantari
Savita
S
Lou
Hong
H
Loh
Y Peng
YP
D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000097573:Red Fluorescent Protein
D043423:Carboxypeptidase H; D002268:Carboxypeptidases; D008164:Luminescent Proteins; D011333:Pro-Opiomelanocortin; D021382:Protein Sorting Signals; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein