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Identification of a Novel Prohormone Sorting Signal-Binding Site on Carboxypeptidase E, a Regulated Secretory Pathway-Sorting Receptor

Section on Cellular Neurobiology (C.-F.Z., Y.P.L.) Laboratory of Developmental Neurobiology National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
Novartis Institute for Medical Research (C.R.S.) London, WCIE 6BN, United Kingdom

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp₁₀,Glu₁₄) and two hydrophobic (Leu₁₁, Leu₁₈) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg₂₅₅ and Lys₂₆₀, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg₂₅₅ or Lys₂₆₀, or both residues substituted, showed no binding of [¹²⁵I]N-POMC_1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg₁₄₇ to Ala₁₄₇ at a substrate-binding site, Arg ₂₅₉ to Ala₂₅₉ and Ser₂₀₂ to Pro₂₀₂, in CPE did not affect the level of [¹²⁵I]N-POMC_1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp₁₀, Leu₁₁, Glu₁₄, Leu₁₈) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg₂₅₅ and Lys₂₆₀ at a novel binding site, independent of the active site on CPE.

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