THE INTEGRITY OF THE RRGDL SEQUENCE OF THE PROPROTEIN CONVERTASE PC1 IS CRITICAL FOR ITS ZYMOGEN AND C-TERMINAL PROCESSING AND FOR ITS CELLULAR TRAFFICKING

In order to define the functional importance of the conserved RRGDL mo tif in the P-domain of the mammalian proprotein convertases (PCs) we g enerated and cellularly expressed three mutant PCl vaccinia-virus (VV) recombinants: ARGDL-PCl RAGDL-PCl and RRGEL-PCl. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demo nstrated that, in part, this effect was due to both an increased degra dation of the mutant PC is within the endoplasmic reticulum and to a d iminished level of zymogen processing in the same compartment. In addi tion, within cells containing secretory granules such as PCl2 and GH4C l cells, such mutations prevented the C-terminal auto-processing of PC I into the fully mature 66 kDa form stored in the secretory granules o f regulated cells. Since the 66 kDa PCI is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the ge neration of maximal intracellular PCI activity. In regulated cells, co -expression of POMC with PCI or its mutants together with the general PC inhibitor alpha(1)-antitrypsin Portland (alpha(1)-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PCl muta nts, whereas it only partially inhibited the ability of wild-type PCl to process POMC. This suggests that RRGDL mutations prevent PCI from e ntering secretory granules and hence the formation of the 66 kDa PCI, and result in the mis-sorting of PCI mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocyto chemical data demonstrating that RRGDL mutants exhibit an intracellula r localization pattern different from that of the granule-associated w ild-type PCl, but similar to that of the Golgi-localized convertase PC 5-B.