Tissue-specific processing of neurotensin/neuromedin-N precursor in cat

Antisera were raised towards the putative N-terminal sequence of the canine precursor to neurotensin (NT) and neuromedin N (NMN), residues 24-35 in the pre-prohormone, as well as its C-terminal TAIL, residues 164-170. These were used in conjunction with previously developed immunoassays towards NT and NMN to characterize the precursor and to study its processing in feline brain, heart, adrenal and intestine. The precursor was identified as a 18 kDa protein that reacted with both the N- and C-terminal antisera and yielded NT3-13 and NT4-13 upon treatment with pepsin. It represented 5-10% of the NT activity in ileum. Processing was found to be tissue-specific, yielding primarily NT, NMN and TAIL in most tissues but also giving rise to N-terminally extended forms of these peptides, which were particularly evident in extracts of adrenal and intestine. Two of the four molecular forms detected using the TAIL immunoassay were tenatively identified as NT-TAIL and Gly-Ser-Tyr-Tyr-Tyr based upon chromatographic evidence. Western blotting indicated that ileal extracts contained large molecular forms of NT (17 kDa) and NMN (16 kDa), whereas brain extracts contained primarily the N-terminal of the precursor without NT and NMN attached (14.3 kDa). It was concluded that the structure of the feline precursor in its N- and C-terminal regions, and the positioning of the signal peptide cleavage site were as predicted from cDNA work in the dog. On the other hand, processing of the precursor was complex, occurring in a tissue-specific manner.