Abstract
Many peptide hormones are produced from larger precursors by endoproteolysis at pairs of basic amino acids (e.g. Lys-Arg and Arg-Arg) within the regulated secretory pathway in endocrine cells. However, many other secretory and membrane proteins appear to be produced from precursors through cleavage at multiple, rather than paired, basic residues within the constitutive secretory pathway in non-endocrine cells. By surveying various precursors processed constitutively, we noticed that most of them have the consensus sequence, Arg-X-Lys/Arg-Arg (RXK/RR), at the cleavage site. When expressed in endocrine and non-endocrine cells, a precursor with the RXKR sequence was cleaved in both types of cells, whereas that with the Lys-Arg pair was cleaved only in the endocrine cells. When the RXKR precursor was coexpressed with furin and PC3, both of which are mammalian homologues of the yeast precursor-processing endoprotease Kex2, in non-endocrine cells, enhancement of the precursor cleavage by furin but not by PC3 was observed. By contrast, when the Lys-Arg precursor was coexpressed with the two mammalian proteases in endocrine cells with no endogenous processing activity at dibasic sites, it was cleaved only by PC3. These results indicate that the basic pair and the RXK/RR sequence are the signals for precursor cleavages catalyzed by PC3 within the regulated secretory pathway and by furin within the constitutive pathway, respectively.
Highlights
EXPERIMENTAL PROCEDURESAtT-20 cell poly(A)+ RNA using the cDNA synthesissystem (Amersham Corp.) and inserted into the EcoRI site of the XgtlO vector
Endoproteolytic precursor cleavage is one of the key steps t o yield bioactive peptides
There have been several reports on endoprotease activities capable of precursor cleavage [4,5,6,7,8,9],little is known about endoproteases physiologically involved in this process
Summary
AtT-20 cell poly(A)+ RNA using the cDNA synthesissystem (Amersham Corp.) and inserted into the EcoRI site of the XgtlO vector. Phe residue atthefourth residue upstream of the cleavage site (position -4) of mouse Ren-2 prorenin by Arg was performed using the primer shown in Fig. 2A in a previously described manner [22]. CDNA fragments covering the entire coding sequence of native and mutant prorenins, furin [17], and PC3 were subcloned behind the SV40 promoter of the pSVD vector [23]. Stable transfectants of CHO [23] and AtT-20 [24] cells were selected under previously described conditions. CHO or GH,C1 cells a t -70% confluence in a 35-mm dish were transfected with thexpression plasmid of native or mutant prorenin in combination with thatof furin or PC3, incubatedfor 48 h, and used for the following experiments. The abbreviations used are: POMC, pro-opiomelanocortin; CHO, Chinese hamster ovary; NDV, Newcastle disease virus; HIV, human immunodeficiency virus; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 8-Br-cAMP, 8-bromo-CAMP
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