7B2 Is a Specific Intracellular Binding Protein of the Prohormone Convertase PC2

Abstract: Biosynthetic pulse‐chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro‐PC2 and that zymogen activation to PC2 occurs following the slow exit of pro‐PC2 from the endoplasmic reticulum (ER) and its concentration within the trans ‐Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro‐7B2), which is cleaved within the TGN by a furin‐like ubiquitous convertase at the RRKRR 155 S site to generate 7B2. In this report, we demonstrate that within the ER, pro‐7B2 binds pro‐PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca 2+ dependent and does not require an N ‐glycosylated pro‐PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR 152 and greatly diminished by mutations of either the R 151 or S 156 residues of pro‐7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin‐like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca 2+ ‐dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro‐PC2, did not eliminate the processing of pro‐PC2 to PC2. Accordingly, cellular coexpression of 7B2 together with PC2 and proopiomelanocortin only marginally diminished the ability of PC2 to cleave proopiomelanocortin into β‐endorphin in constitutive cells and had no effect in regulated cells. These results suggest that in vivo pro‐7B2 is a specific PC2‐binding protein that only transiently inhibits the processing of pro‐PC2 until it reaches the TGN.