Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat—I. Biochemical characterization of maturation products

Abstract

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions. This lower content was not the consequence of a lack of precursor processing at the dibasic site that flanks the N-terminus of neuromedin N but, rather, resulted from atypical cleavage of this site or from differential post-processing disposal of neuromedin N. The high levels of precursor messenger RNA previously reported by others in the hippocampus relative to other brain regions were not matched by high concentrations of precursor-derived products. In conclusion, the immunological and biochemical approaches described here provide powerful means for establishing the processing pattern of the neurotensin/neuromedin N precursor in brain regions. The present data set the biochemical basis for the immunohistochemical studies on the regional and cellular brain distribution of neurotensin/neuromedin N precursor-derived peptides, as reported in a companion paper.