Peptidyl-glycine alpha-amidating monooxygenase targeting and shaping of atrial secretory vesicles: inhibition by mutated N-terminal ProANP and PBA.
ANP (atrial natriuretic peptide) is widely recognized as an important vasorelaxant, diuretic, and cardioprotective hormone. Little is known, however, about how ANP-secretory vesicles form within the atrial myocytes. Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing proANP-enhanced green fluorescent protein (EGFP), or N-terminal-mutated fusion proteins thought to suppress the calcium-dependent aggregation of proANP. Results showed the following: (1) aggregates of proANP and coexpressed proANP-EGFP recruited peptidylglycine alpha-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal-mutated (Glu23,24-->Gln23,24) and N-terminal-deleted proANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 mumol/L), a pharmacological inhibitor of the lumenal peptidylglycine alpha-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP-PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking. These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal proANP and on the lumenal domain of PAM-1. Conversely, PAM-1 participates in shaping the proANP-secretory vesicles. The full text of this article is available online at http://circres.ahajournals.org.
Labrador
Vénus
V
Department of Neuroscience, Centre Médical Universitaire, University of Geneva, Switzerland.
Brun
Cécile
C
König
Stéphane
S
Roatti
Angela
A
Baertschi
Alex J
AJ
D000818:Animals; D020517:Atrial Appendage / Q000378:metabolism* / Q000648:ultrastructure; D009320:Atrial Natriuretic Factor / Q000502:physiology*; D001665:Binding Sites; D018274:Electroporation; D005089:Exocytosis / Q000187:drug effects; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000502:physiology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D024661:Potassium Channels, Inwardly Rectifying / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000502:physiology; D022142:Secretory Vesicles / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D005229:Fatty Acids, Monounsaturated; C489461:Kir2.1 channel; D009097:Multienzyme Complexes; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C064180:4-phenyl-3-butenoic acid; D009320:Atrial Natriuretic Factor; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase