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 RRKRR155S 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 Ca2+ 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 RR152 and greatly diminished by mutations of either the R151 or S156 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.(ABSTRACT TRUNCATED AT 250 WORDS)