Discrimination between temperature- and brefeldin A-sensitive steps in the sulfation, phosphorylation, and cleavage of progastrin and its derivatives.
Maturation of the acid-stimulating hormone gastrin involves precursor cleavage, tyrosine sulfation, serine phosphorylation, and COOH-terminal amidation. We have used brefeldin A and incubation at 22 degrees C to determine where and when these modifications occur. Immunogold studies of gastrin cells incubated at 22 degrees C revealed swollen Golgi cisternae, the terminal regions of which were associated with an accumulation of progastrin immunoreactivity. At 22 degrees C, [3H]tyrosine and [35S]sulfate were incorporated into progastrin, but Arg94-Arg95 cleavage, and Ser96 phosphorylation, were inhibited. When pulse labeling at 22 degrees C for 120 min was followed by a chase at 37 degrees C, [35S]progastrin was cleaved at Arg94-Arg95 with a t1/2 of about 10 min, compared with about 20 min for [3H]progastrin. Approximately 60% of the COOH-terminal cleavage fragment was phosphorylated, but there was little or no incorporation of [32P]phosphate into progastrin. Addition of brefeldin A during the chase substantially inhibited cleavage of [3H]progastrin, but not [35S]progastrin. However, when pulse labeling was limited to 20 min at 22 degrees C, the presence of brefeldin A in a subsequent chase at 37 degrees C completely inhibited cleavage of [35S]progastrin. The data indicate that progastrin sulfation occurs in the trans-Golgi network, exit from which involves passage through first a brefeldin A-sensitive and then a temperature-sensitive step. Cleavage at Arg94-Arg95 and Ser phosphorylation are closely linked, occur distal to the temperature-sensitive step, and are followed by amidation in secretory granules. It is known that mature secretory granules do not phosphorylate progastrin-derived peptides, and so phosphorylation appears to coincides with, and may provide a marker for, delivery of peptide from trans-Golgi work to immature secretory granules in gastrin cells.
Varro
A
A
Physiological Laboratory, University of Liverpool, United Kingdom.
Henry
J
J
Vaillant
C
C
Dockray
G J
GJ
D000577:Amides / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A; D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000494:pharmacology*; D005755:Gastrins / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D013431:Sulfates / Q000378:metabolism*
D000577:Amides; D003517:Cyclopentanes; D005755:Gastrins; D011498:Protein Precursors; D013431:Sulfates; D020126:Brefeldin A; C013706:big gastrin