5-HT1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT1B or 5-HT1D receptors, it was shown that 5-HT-moduline prevents the binding of [3H]5-HT to 5-HT1B as well as to 5-HT1D receptors with similar biochemical characteristics: the IC50 of the peptide was 1.2x10(-12) M for 5-HT1B and 9x10(-13) M for 5-HT1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [3H]5-HT on 5-HT1B (-51.2 +/- 1%) as well as 5-HT1D binding (-47.2 +/- 7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the "scrambled" peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT1A or 5-ht6 receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [35S]GTPgammaS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC50 = 1.2 +/- 0.7x10(-12) M for 5-HT1B and 9.8 +/- 4.0x10(-12) M for 5-HT1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT1D as well as 5-HT1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT1B and 5-HT1D receptors.
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 ...
The serotonergic transmission is considered as a neuromodulatory system in the Central Nervous System. 5-HT1B receptors play an important role in this modulatory activity. We have purified from mammalian brain an endogenous peptide, LSAL, we called 5-HT-moduline, interacting specifically with 5-HT1B receptors. This interaction is characterized by a high affinity (Ki = 10(-10) M) and a non-competitive mechanism. Direct [3H]5-HT-moduline binding revealed a single population of sites having an apparent affinity constant close to 10(-10) M. Autoradiographic studies showed a brain distribution of [3H]5-HT-moduline binding sites closely related to the 5-HT1B receptors. In functional studies, the peptide is able to reverse the activity of a 5-HT1B agonist in the nanomolar range. Furthermore, this antagonist effect is also observed in vivo on mice behavior. Immunocytochemistry revealed an heterogeneous distribution of 5-HT-moduline in mouse brain. The labeled structures correspond to cellular profiles with axon-like prolongations. Moreover, in vitro, LSAL is released in a Ca++, K(+)-dependent manner. Therefore, 5-HT-moduline behaves as a neurotransmitter. The fact that 5-HT-moduline induces the desensitization of 5-HT1B receptors reflects the existence of a novel and efficient mechanism able to rapidly modulate the serotonergic activity.
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