Testis-specific prohormone convertase PC4 processes the precursor of pituitary adenylate cyclase-activating polypeptide (PACAP).

The physiological substrate for proprotein convertase (PC) 4, which is expressed only in the testis, has remained unknown. Pituitary adenylate cyclase activating polypeptide (PACAP), originally isolated from the hypothalamus, exists as two amidated forms with 38 (PACAP38) and 27 (PACAP27) residues. PACAP-like immunoreactivity (PACAP-li) is found not only in the brain, but also in the peripheral tissues, and is especially abundant in the testis. Immunohistochemistry of the rat testis demonstrated strong PACAP-li in spermatids in the cap and acrosome phases. The nearly simultaneous expression of PC4 transcripts and PACAP-li in spermatids during spermatogenesis led to the hypothesis that PACAP precursor is processed by PC4. To investigate this possibility, rat pituitary GH4C1 cells were stably transfected with human PACAP cDNA, and some of these cells were co-transfected with mouse PC4 cDNA. The acid extracts of the cells were fractionated by reversed-phase HPLC. Each fraction was examined for PACAP-li using three antisera which recognize PACAP precursor, PACAP38 and/or PACAP27. Negligible PACAP-li that eluted with synthetic PACAP38 or PACAP27 was detected from cells transfected with PACAP cDNA; however, PC4 co-transfected cells showed marked PACAP-li peaks with the retention times for both PACAP38 and PACAP27. Moreover, Western blot analysis revealed immunostained bands, corresponding to the Mr for PACAP38 and PACAP27, in the PC4 co-transfected cells. Bioactivity, as indicated by stimulation of cAMP production in pituitary cell cultures, was found only in the extracts of PC4 co-transfected cells. These results provide evidence that PACAP precursor in the testis is a substrate for PC4. The processing of PACAP precursor by PC4 at a critical time in spermatogenesis suggests an important regulatory role of PC4 and PACAP in the maturation of germ cells in the testis.