RESUMO
The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.
Assuntos
Proteínas de Membrana/fisiologia , Pró-Proteína Convertase 5/metabolismo , Serina Endopeptidases/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Cisteína , Heparitina Sulfato/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Camundongos , Pró-Proteína Convertase 5/fisiologia , Pró-Proteína Convertases , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/fisiologia , Serina Endopeptidases/fisiologia , TransfecçãoRESUMO
Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them.