Title

Multiple roles for the cytosolic domain of peptidylglycine alpha-amidating monooxygenase (PAM)

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Cell|Chemistry, Biochemistry

Degree

D.N.P.

Abstract

Neuropeptides play an essential role in maintaining homeostasis. Therefore a thorough understanding of the mechanisms involved in the synthesis, trafficking, stimulated secretion and feedback regulation of secretory granules that store neuropeptides is crucial. Peptidylglycine alpha-amidating monooxygenase (PAM), an integral protein of the secretory granule membrane, catalyzes neuropeptide amidation, a modification essential for bioactivity. Targeted deletion of PAM causes embryonic lethality. Mice with a single copy of PAM exhibit increased anxiety-like behavior and seizure sensitivity but only minor deficits in peptide amidation, suggesting additional regulatory roles for PAM in secretory granule function. ^ PAM is composed of a bifunctional enzymatic domain, a transmembrane domain and a cytosolic domain. The objective of this study was to understand the roles of the PAM cytosolic domain. PAM, one of the few known secretory granule membrane proteins, has served as a model to understand granule trafficking as well as a candidate feedback regulator. We demonstrated that the PAM cytosolic domain is intrinsically unstructured. Two-dimensional gel electrophoresis revealed the occurrence of coupled phosphorylation and dephosphorylation of this domain during stimulated secretion. Six phosphorylation sites were identified using mass-spectrometry and phospho-specific antibodies. Electron-microscopy and surface biotinylation revealed that dephosphorylation of Ser949 was essential for sorting in the endocytic pathway, juxtamembrane cleavage of PAM and returning PAM to granules. Stimulated exocytosis increased the production of sf-CD, a soluble, cytosolic fragment of PAM that accumulated in the nucleus. Prior cleavage of PAM by prohormone convertases in the granule pathway or by α-secretase in the non-granule pathway was required before intramembrane proteolysis could be catalyzed by γ-secretase. Cytosolic domain phosphorylation affected the production of sf-CD and mass spectroscopy identified sf-CD that included 13, 11 or 8 transmembrane domain residues. Taken together, our data support a role for sf-CD in coordinating secretory granule biogenesis. ^