The RGD motif and the C-terminal segment of proprotein convertase 1 are critical for its cellular trafficking but not for its intracellular binding to integrin alpha5beta1.
Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha5beta1 associates primarily with the zymogens proPC1, proPC1-DeltaC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha5beta1.
Rovère
C
C
Laboratories of Biochemical and Molecular Neuroendocrinology, the Protein Engineering Network of Centres of Excellence, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7 Canada.
Luis
J
J
Lissitzky
J C
JC
Basak
A
A
Marvaldi
J
J
Chrétien
M
M
Seidah
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation; D007150:Immunohistochemistry; D009842:Oligopeptides / Q000378:metabolism*; D043484:Proprotein Convertases; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry; D017469:Receptors, Fibronectin / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D009842:Oligopeptides; D016271:Proto-Oncogene Proteins c-myc; D017469:Receptors, Fibronectin; C047981:arginyl-glycyl-aspartic acid; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases