Abstract
Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.
Highlights
Secretory granule membrane proteins are retrieved and reused or degraded after exocytosis
Band intensities were quantified using GeneTools software and exposures in the linear range for quantification. His Cluster Imparts pH Sensitivity to Non-catalytic peptidylglycine ␣-amidating monooxygenase (PAM) Linker Region—The protease-resistant catalytic core of peptides through its hydroxylation domain (PHM) (PHMcc) is followed by a well conserved cluster of three His residues (Fig. 1, A and B). This His cluster is included in the final exon encoding PHMcc and is followed by a poorly conserved, protease-sensitive region encoded by a short exon present in each of the major splice variants of PAM
The non-catalytic linker region between PHM and PAL can end at exon 15, as in PAM-2, or can include a 315-nt exon, as in PAM-1 (Fig. 1, A and B) [42]
Summary
Secretory granule membrane proteins are retrieved and reused or degraded after exocytosis. Conclusion: Lacking this conserved His cluster, proteolytic maturation and generation of a soluble cytosolic fragment of amidating enzyme fail to occur, disrupting secretion and nuclear signaling. The two catalytic domains of peptidylglycine ␣-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-HisHis) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wildtype AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue
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