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High‐Affinity Receptor Sites and Rapid Proteolytic Inactivation of Neurotensin in Primary Cultured Neurons
Author(s) -
Checler Frédéric,
Mazella Jean,
Kitabgi Patrick,
Vincent JeanPierre
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb13083.x
Subject(s) - neurotensin , endopeptidase , receptor , peptide , biochemistry , cleavage (geology) , chemistry , neuropeptide , biology , dissociation constant , enzyme , paleontology , fracture (geology)
The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37°C of [ monoiodo ‐Tyr 3 neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 p M and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preprations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro 7 ‐Arg 8 , Arg 8 ‐Arg 9 , and Pro 10 ‐Tyr 11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro 7 ‐Arg 8 bond and contributes to the hydrolysis mainly at the Pro 10 ‐Tyr 11 site. However, the latter breakdown is also generated by a neurotensin‐degrading neutral metallopeptidase. The cleavage at the Arg 8 ‐Arg 9 bond is due to a peptidase that can be specifically inhibited by N ‐[1 ( R, S )‐carboxy‐2‐phenylethyl]‐alanyl‐alanyl‐phenylalanyl‐ p ‐aminobenzoate. The secondary processing occurring on neurotensin degradation products are: (1) a bestatin‐sensitive aminopeptidasic conversion of neurotensin, 11‐13 to free Tyr 11 , and (2) a rapid cleavage of neurotensin 8‐13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.