title: A histidine-rich linker region in peptidylglycine α-amidating monooxygenase has the properties of a pH sensor.
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.
authors.affiliation: From the Departments of Neuroscience and.
authors.forename: Kurutihalli
authors.initials: K
authors.lastname: Vishwanatha
authors.forename: Nils
authors.initials: N
authors.lastname: Bäck
authors.forename: Richard E
authors.initials: RE
authors.lastname: Mains
authors.forename: Betty A
authors.initials: BA
authors.lastname: Eipper
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D004705:Endocytosis / Q000235:genetics; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008854:Microscopy, Electron; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D059748:Proteolysis; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D021601:trans-Golgi Network / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D009097:Multienzyme Complexes; D020033:Protein Isoforms; D006639:Histidine; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase
keywords: Endocytosis; Glycoprotein Secretion; Neuropeptide; Protein Secretion; pH Regulation