Furin and prohormone convertase 1/3 are major convertases in the processing of mouse pro-growth hormone-releasing hormone.
We investigated the proteolytic processing of mouse pro-GHRH [84 amino acids (aa)] by furin, PC1/3, PC2, and PC5/6A. We created six point mutations in the N- and C-terminal cleavage sites, RXXR decreased and RXRXXR decreased, respectively. The following results were obtained after transient transfection/cotransfection and metabolic pulse-chase labeling studies in several neuroendocrine cells. 1) Furin was the most efficient convertase in cleaving the N-terminal RXXR/RXRR site to generate intermediate I, 12-84aa, whereas PC1/3 was the most potent in processing the C-terminal RXRXXR site to yield mature GHRH, 12-53aa. 2) Both PC1/3 and PC5/6A also processed the N-terminal site but less efficiently than furin. 3) PC2 was much weaker in cleaving the C-terminal site relative to PC1/3 to generate mature GHRH. 4) The Q10R mutant was significantly more susceptible to furin cleavage at the N-terminal site than the wild-type pro-GHRH. And 5) the N- and C-terminal P1 Arg residues, R11 and R54, respectively, were essential for mature GHRH production. We also showed localization of the GHRH immunoreactive peptides in Golgi and secretory granules in neuroendocrine cells by an immunofluorescence assay. We conclude that the efficient production of mature GHRH from pro-GHRH is a stepwise process mediated predominantly by furin at the N-terminal cleavage site followed by PC1/3 at the C terminus.
Dey
Arunangsu
A
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
Norrbom
Christina
C
Zhu
Xiaorong
X
Stein
Jeffrey
J
Zhang
Chunling
C
Ueda
Kazuya
K
Steiner
Donald F
DF
D000818:Animals; D002460:Cell Line; D045683:Furin / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D045664:Proprotein Convertase 1 / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D010446:Peptide Fragments; D011498:Protein Precursors; C062302:pro-growth hormone releasing hormone, mouse; D013007:Growth Hormone-Releasing Hormone; D045683:Furin; D045664:Proprotein Convertase 1