5-Hydroxytryptamine-moduline is an endogenous cerebral tetrapeptide that regulates the activity of 5-hydroxytryptamine 1B receptors. Direct binding of 5-[ 3 H]hydroxytryptamine-moduline on rat brain homogenate evidenced the existence of two interacting sites for the peptide, very likely corresponding to different conformations of the 5-hydroxytryptamine 1B receptor: The peptide first binds to a low-affinity state of the receptor (pIC 50 ϭ 7.68 Ϯ 0.14) and then induces (or stabilizes) a high-affinity complex (pIC 50 ϭ 11.62 Ϯ 0.18). This work focuses on the ability of 5-hydroxytryptamine-moduline analogues to recognize the high-and low-affinity sites for 5-hydroxytryptamine-moduline. The results obtained show that the two conformers of the 5-hydroxytryptamine 1B receptor have similar but not identical binding pockets for 5-hydroxytryptamine-moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low-affinity site than the high-affinity one, which only needs a short hydrophobic amino acid in position 2. Key Words: 5-Hydroxytryptaminemoduline-5-Hydroxytryptamine receptor-Binding-Structure-activity relationships. J. Neurochem. 73, 2617Neurochem. 73, -2620Neurochem. 73, (1999.5-Hydroxytryptamine (5-HT)-moduline (LSAL) is an endogenous peptide modulating the serotonergic system activity via its specific interaction with the 5-HT 1B receptors (Massot et al., 1996). The peptide binds directly to the 5-HT 1B receptor protein and inhibits in a noncompetitive manner the binding of [ 3 H]5-HT to the receptor and its activity.It was recently shown (M. Plantefol et al., submitted) that [ 3 H]5-HT-moduline binding was cooperative and heterogeneous. The two binding affinities (pIC 50 ϭ 11.62 Ϯ 0.18 and 7.68 Ϯ 0.14) are related to two interconvertible conformations of the receptor, which will be called, respectively, the highaffinity and low-affinity conformation of the receptor (for the peptide). It was also shown that 5-HT-moduline first binds to the low-affinity conformer, slowly inducing or stabilizing the high-affinity complex between the receptor and the peptide.As the induction kinetics of the high-affinity conformer are slow, it was possible to use an incubation procedure that evidenced the binding of [ 3 H]5-HT-moduline to both conformations of the receptor via the competition curves of LSAL with the tritiated peptide. The first goal of this work was to study the structural requirements of a series of 5-HT-modulinederived peptides to interact with the 5-HT 1B receptor. For that purpose, 27 analogues were constructed from the sequence of 5-HT-moduline, including changes of one or more amino acids with either natural or unnatural amino acids. All these analogues were tested in binding competition assays to determine their capacity ...
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