Processing of proinsulin by transfected hepatoma (FAO) cells.
Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.
Vollenweider
F
F
Laboratoires de Recherche Louis Jeantet, University of Geneva, Switzerland.
Irminger
J C
JC
Gross
D J
DJ
Villa-Komaroff
L
L
Halban
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D003907:Dexamethasone / Q000494:pharmacology; D016521:Electrophoresis, Gel, Pulsed-Field; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011384:Proinsulin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D003907:Dexamethasone; D004247:DNA; D011384:Proinsulin