Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors

doi:10.1016/S0196-9781(03)00064-0

Peptides

Volume 24, Issue 3, March 2003, Pages 469-475

https://doi.org/10.1016/S0196-9781(03)00064-0 Get rights and content

Abstract

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.

Introduction

The tachykinins are a group of neuropeptides characterized by the conserved C-terminal sequence, Phe–Xaa–Gly–Leu–Met–NH₂ (Fig. 1). Substance P (SP) is the most well studied tachykinin and has been implicated in a number of physiological roles including inflammation, nociception, and regulation of smooth muscle. In addition to SP, several tachykinin peptides have been found to be active in invertebrates and in nonmammalian vertebrates, particularly amphibians [10], [11]. Three such peptides, ranatachykinin A (RTKA), ranatachykinin B (RTKB) and ranatachykinin C (RTKC), have been isolated from bullfrog (Rana catesbeiana) brain and gut [7].

To date the actions of the ranatachykinins (RTKs) on neuronal cells have not been studied. The RTKs actions were characterized using the classical pharmacology preparations of rat duodenum and guinea pig ileum [7]. Another study of several tachykinins, including RTKA, looked at peptide action on ion transport in the frog skin mediated by a neurokinin-1-like receptor [9]. We have previously reported the effects of the RTKs to produce Ca²⁺ elevations and receptor desensitization at the bullfrog substance P receptor (bfSPR) in a heterologous expression system [13]. In the present study, we have assessed the effects of the RTKs to modulate the M-current of sympathetic neurons from the bullfrog. This provides the first description of actions of the RTK peptides on native neurons.

The cDNA for the bfSPR is an orthologue of the mammalian neurokinin 1 receptor (NK1R) based on structural identity and ligand binding characteristics [16]. The rat (r)NK1R and bfSPR are 69% identical at the amino acid level, with the largest degrees of conservation seen in the transmembrane and intracellular loop regions. Nine of the ten proposed binding interactions between SP and the rNK1R are completely conserved in the bfSPR and the remaining binding site is likely conserved due to tertiary structural conditions caused by a disulfide bond in the extracellular domain.

In addition to characterizing the actions of the RTKs on neurons, we have correlated peptide binding properties and receptor activation with the ability of the peptide to cause receptor desensitization. Our lab has previously shown that a tachykinin’s ability to produce signaling is not directly proportional to its ability to produce desensitization [13], but it is not known if these differences are due to differences in binding properties of the RTKs, if these effects are the same in neurons as in the heterologous expression system or if these properties are specific for the bfSPR. In the present study we have examined the binding properties, signal activation and receptor regulation properties of four tachykinin peptides, SP, RTKA, RTKB and RTKC, at the bfSPR and rNK1R.

Section snippets

Whole cell electrophysiology

Single neurons were dissociated from bullfrog sympathetic ganglia [17]. Briefly, ganglia were dissected, treated with enzymes and stored in growth medium at 4 °C for 1–3 days. The ganglia were triturated to release single neurons for daily use. Under these conditions synaptic contacts do not form between isolated neurons.

Whole cell recordings of isolated neurons were conducted at room temperature, ∼23 °C, with a patch clamp amplifier (Warner PC-501, Hamden, CT). Peptides were dissolved in

Peptide inhibition of M-type potassium current

The effects of RTKA, RTKB and RTKC on native neurons have been assessed by measuring the change in I_M following application of the peptides at various concentrations. Each of the RTKs resulted in an inhibition of I_M. The concentration–response curves for inhibition of I_M are presented in Fig. 3. Like SP, the RTKs inhibit I_M in a concentration-dependent manner ( $F(8,69)=3.14$ , $P<0.005$ ) with similar maximum effects. The rank order of potency for inhibiting I_M is $RTKA > SP > RTKC = RTKB$ with EC₅₀ values

Discussion

Two major findings are presented here. First, the RTKs affect single bullfrog neurons by inhibiting the M-current. This is the first evidence that the RTKs regulate neuronal excitability by activating an endogenous tachykinin receptor. Second, we have studied the correlation between receptor binding, signal activation and the receptor regulation of desensitization and internalization. There is no direct correlation of a peptide’s ability to bind to the NK1R and activate second messenger

Conclusion

We have shown that the RTKs act as full agonists at an endogenous tachykinin receptor expressed in sympathetic ganglia neurons from the bullfrog. This interaction results in inhibition of a potassium current known as the M-current and likely causes neuronal excitability to be altered and fine-tuned. This response undergoes rapid desensitization thereby giving another mean of regulating cellular activity. These findings show that the RTKs, which were isolated from bullfrog brain and gut, act at

Acknowledgements

We would like to thank Dr. Gary Meszaros, Dr. James Krause and Ms. Anne Shriner for their assistance and contributions.

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Cited by (4)

The role of the N-terminal and mid-region residues of substance P in regulating functional selectivity at the tachykinin NK<inf>1</inf> receptor
2008, European Journal of Pharmacology
Previous studies have shown that tachykinin peptide ligands of the tachykinin NK₁ receptor exhibit functional selectivity with respect to signal activation and desensitization. The differences are most dramatic between the naturally occurring peptides substance P (RPKPQQFFGLM-NH₂) and ranatachykinin C (HNPASFIGLM-NH₂). To understand the structural features of the peptides that underlie these differences, four peptide analogs have been designed and tested. The analogs were designed to assess the major structural differences between substance P and ranatachykinin C, including the role of the N-terminal Arg and the substitution of the mid-region Glns with Ala and Ser (Q5 replaced with A and/or Q6 replaced with S). Receptor binding, receptor activation of intracellular calcium fluxes, and receptor desensitization of the rat tachykinin NK₁ receptor were quantified for each ligand. All of the peptides bound to the rat tachykinin NK₁ receptor with high affinity, produced robust calcium signal activation, and led to agonist-induced receptor desensitization. It was found that deletion of the N-terminal Arg of substance P or replacement of either or both Q5 and Q6 altered the functional selectivity of substance P based on the relationship of receptor binding to receptor activation and activation to desensitization. When considered in light of our previously published nuclear magnetic resonance structure data, the data presented herein suggest that the one, five and six positions of the substance P backbone are key structural residues that govern the relative degree of tachykinin peptide-mediated receptor signaling and desensitization.
Delineation of the motilin domain involved in desensitization and internalization of the motilin receptor by using full and partial antagonists
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Citation Excerpt :
From these data we can conclude that the ability of a peptide to induce desensitization is not solely determined by its ability to act as an agonist for signal activation events. Disparities between activation and desensitization have also been described for dopamine D1A receptors [29], μ-opioid receptors [30], the human tachykinin NK-1 [31] and serotonin 5-HT2C receptors [32]. Fusion of an EGFP protein to the carboxyl terminus of the MTLR did not seem to alter the pharmacological and functional properties of the MTLR.
Studies with fragments of the gastrointestinal peptide, motilin, indicate that the C-terminal region of this peptide plays an important role in the desensitization of the motilin receptor (MTLR).
To verify this hypothesis we studied the desensitization, phosphorylation and internalization induced by motilin analogues of different chain length with agonistic and antagonistic properties in CHO–MTLR cells.
We studied motilin [1–22], the [1–14] fragment, the analogues Phe³[1–22] and Phe³[1–14], and two putative antagonists, GM-109 and MA-2029 (modified 1–4 and 1–3 fragments). Activation and desensitization (2 h preincubation with the motilin analogues 10 μM) were studied in CHO–MTLR cells by an aequorin based luminescence assay. Phosphorylation was studied by immunoprecipitation and internalization was visualized in CHO–MTLR cells containing an enhanced green fluorescent protein (CHO–MTLR–EGFP).
Motilin [1–22] and [1–14] were more potent than Phe³[1–22] and Phe³[1–14] (pEC₅₀: 9.77, 8.78, 7.36 and 6.65, respectively) to induce Ca²⁺ release. GM-109 and MA-2029 were without agonist activity. [1–22] and Phe³[1–22] decreased the second response to motilin from 78 ± 2% to 11 ± 3% and 34 ± 3% (P < 0.001), respectively, whereas [1–14], Phe³[1–14], GM-109 and MA-2029 had no desensitizing effect (68 ± 5%, 78 ± 3%, 78 ± 6% and 78 ± 5%, respectively, P > 0.05). The rank order of MTLR-phosphorylation was: [1–22] > [1–14] > Phe³[1–22] = Phe³[1–14] > GM-109 = MA-2029. Only motilin [1–22] and [1–14] induced receptor MTLR–EGFP internalization as shown by a decrease in membrane fluorescence: 20 ± 3% and 7 ± 3%, respectively.
The C-terminus of motilin enhances desensitization, phosphorylation and internalization of the MTLR while modifications of the N-terminus can favor a conformation of the receptor that is less susceptible to phosphorylation and internalization.
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^☆: This work was supported by NIH grant NS25999 from the National Institute of Neurological Disorders and Stroke to MAS.

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Peptides

Abstract

Introduction

Section snippets

Whole cell electrophysiology

Peptide inhibition of M-type potassium current

Discussion

Conclusion

Acknowledgements

References (20)

Partial agonists and G protein-coupled receptor desensitization

Trends Pharmacol. Sci.

Four novel tachykinins in frog (Rana catesbeiana) brain and intestine

Regul. Pept.

Actions of tachykinins on the ion transport across the frog skin

Peptides

The mammalian tachykinin receptors

Gen. Pharmacol.

Desensitization of the inhibition of the M-current in sympathetic neurons: effects of ATP analogs polyanions and multiple agonist applications

Neuron

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana

Neuroscience

The N-terminal domain of substance P is required for complete homologous desensitization but not phosphorylation of the rat neurokinin 1 receptor

Neuropeptides

μ-Opioid receptors: ligand-dependent activation of potassium conductance desensitization and internalization

J. Neurosci.

Protein kinase C is not necessary for peptide-induced suppression of M-current or for desensitization of the peptide receptors

Proc. Natl. Acad. Sci. U.S.A.

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells

Biochem. J.

Cited by (4)

The role of the N-terminal and mid-region residues of substance P in regulating functional selectivity at the tachykinin NK<inf>1</inf> receptor

Delineation of the motilin domain involved in desensitization and internalization of the motilin receptor by using full and partial antagonists

Effects of peptides, with emphasis on feeding, pain, and behavior A 5-year (1999-2003) review of publications in Peptides

Functional selectivity of NK<inf>1</inf> receptor signaling: Peptide agonists can preferentially produce receptor activation or desensitization

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors☆

Abstract

Introduction

Section snippets

Whole cell electrophysiology

Peptide inhibition of M-type potassium current

Discussion

Conclusion

Acknowledgements

Trends Pharmacol. Sci.

Regul. Pept.

Peptides

Gen. Pharmacol.

Neuron

Neuroscience

Neuropeptides

μ-Opioid receptors: ligand-dependent activation of potassium conductance desensitization and internalization

J. Neurosci.

Protein kinase C is not necessary for peptide-induced suppression of M-current or for desensitization of the peptide receptors

Proc. Natl. Acad. Sci. U.S.A.

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells

Biochem. J.