UNIPROT:P20366 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20366 (substance P)
21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The neurokinin-1 receptor binds neurokinin peptides with the potency order of substance P > substance K > neurokinin B. Elucidating the molecular basis of differential peptide selectivity will require the localization of the binding domain on the receptor. In the present report, mutagenesis and heterologous expression experiments reveal that a segment of the extracellular N-terminal sequence of the neurokinin-1 receptor is required for the high-affinity binding of substance P and related peptide agonists. Substitution of amino acid residues in the N-terminal region of the receptor affects the binding affinity of both intact peptides and a C-terminal substance P "analog", but not of a nonpeptide antagonist. Glycosylation of the receptor does not change the peptide binding affinity. In addition, substitution of the valine-97 residue in the rat neurokinin-1 receptor by a glutamate residue increases the binding affinity of neurokinin B but not substance P or substance K, suggesting that the second extracellular segment is involved in peptide selectivity. These results indicate that the extracellular domains of neurokinin-1 receptor play a critical role in peptide binding.
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PMID:The extracellular domain of the neurokinin-1 receptor is required for high-affinity binding of peptides. 128 Jan 61

A tachykinin peptide was isolated from an extract of the intestine of the European green frog, Rana ridibunda, and its primary structure was established as: His-Lys-Leu-Asp-Ser-Phe-Ile-Gly-Leu-Met.CONH2. This sequence was confirmed by chemical synthesis and shows two amino acid substitutions (leucine for threonine at position 3 and isoleucine for valine at position 7) compared with neurokinin A. Binding parameters for synthetic [Leu3,Ile7]neurokinin A and mammalian tachykinins were compared using receptor-selective radioligands and crude membranes from tissues enriched in the NK1, NK2 and NK3 receptors. [Leu3,Ile7]Neurokinin A was approx. 3-fold less potent than substance P in inhibiting the binding of 125I-labelled [Sar9,Met(O2)11]substance P (labelled with Bolton-Hunter reagent) to rat submandibular gland (NK1 receptor), 8-fold less potent than neurokinin A in inhibiting the binding of [2-[125I]iodohistidine1]neurokinin A to rat stomach fundus (NK2 receptor) and 6-fold less potent than neurokinin B in inhibiting the binding of 125I-Bolton-Hunter-labelled scyliorhinin II to rat brain (NK3 receptor). Thus the frog neurokinin A-related peptide shows moderate affinity but lack of selectivity for all three tachykinin-binding sites in rat tissues. This non-selectivity is similar to that displayed by the molluscan tachykinin, eledoisin, which also contains an isoleucine residue in the corresponding position in the molecule.
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PMID:Primary structure and receptor-binding properties of a neurokinin A-related peptide from frog gut. 133 83

We have prepared a series of conformationally constrained hexapeptide analogs of substance P which are 500-1500-fold more potent as inhibitors of 125I-labeled Bolton Hunter-conjugated eledoisin binding to rat brain cortex membranes than as inhibitors of 125I-labeled Bolton Hunter-conjugated substance P binding. These analogs stimulate guinea pig ileum contraction (ED50 1-16 nM) and stimulate rat vas deferens contraction (ED50 2-4 microM). However, these peptides are poor stimulators of rat salivation (greater than 40 nmol/100 g body weight). Thus, based on both their receptor potency and pharmacological potency, these peptides are potent and selective tachykinin analogs. These data indicate that a specific carboxyl-terminal conformation is recognized by the 125I-labeled Bolton Hunter-conjugated eledoisin binding site and that this conformation is different from the conformation recognized by the 125I-labeled Bolton Hunter-conjugated substance P binding site. Hexapeptides containing phenylalanine, isoleucine, and valine identical with the carboxyl-terminal sequences of substance P, eledoisin, and neurokinin B, respectively, were nearly equipotent as inhibitors of 125I-labeled Bolton Hunter-conjugated eledoisin binding. The valine analog was only approximately 5-fold less potent than the isoleucine and phenylalanine analogs as an inhibitor of 125I-labeled Bolton Hunter-conjugated substance P binding. Thus, unknown determinants in the amino-terminal sequences of substance P must strongly contribute to the carboxyl-terminal peptide selectivity and conformation. The contraction of guinea pig ileum induced by one of the conformationally constrained analogs is attenuated by pretreatment of the tissue with atropine (2 microM), while that induced by substance P methyl ester, a selective inhibitor of 125I-labeled Bolton Hunter-conjugated substance P binding, is not. Thus, the constrained analog has a higher affinity for the tachykinin receptors in the guinea pig myenteric plexus which are responsible for acetylcholine release than for the tachykinin receptors present on the smooth muscle cells.
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PMID:Conformationally constrained tachykinin analogs which are selective ligands for the eledoisin binding site. 241 47

Isolated rat dorsal root ganglia incorporated [3H]valine, [3H]serine, [3H]phenylalanine and [3H]histidine into a peptide which co-eluted with authentic substance K (neurokinin alpha) on reverse-phase HPLC. Confirmation of the identity of this peptide with substance K was obtained by demonstrating that the sulphoxide derivative of the peptide co-eluted with substance K sulphoxide. Substance P and substance K were synthesised in the ratio 2.6:1. The implications of these results for the co-synthesis of substance P and substance K in sensory neurones are discussed.
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PMID:Rat sensory ganglia incorporate radiolabelled amino acids into substance K (neurokinin alpha) in vitro. 608 8

Four substance P (SP) antagonists were tested on anaesthetized rats, by injecting 8 microgram amounts into the spinal cord (T8-T9), and by observing their effects on the hypothalamo-neurohypophysial responses to a presumably painful stimulus, the superfusion of the hepatic portal vein with 1 microM bradykinin. Only two antagonists, the new D-Pro4,D-Trp7,9,10,Val8-SP4-11 and D-Pro4,D-Trp7,9,10-SP4-11 were capable of inhibiting the responses by 50-60%, the former compound having 3 times less agonistic activity. The results suggest that substitution of the aromatic phenylalanine by the non-polar valine in position 8 may significantly improve the overall characteristics of neurally active SP antagonists.
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PMID:Neural actions of several substance P antagonists in the rat spinal cord. 619 60

During evolution mutations have occurred in peptide receptors that are neutral with respect to binding of the natural peptide ligand but frequently affect the binding of nonpeptide antagonists. By systematically introducing the nonconserved residues from the human neurokinin (NK)-1 receptor into the corresponding rat receptor we have attempted to localize the structural elements that are responsible for 15-76-fold higher affinity of three tachykinin nonpeptide antagonists for the human receptor, compared with the corresponding rat receptor. Surprisingly, exchange of the four divergent residues located around the previously located apparent binding site for CP 96,345 and FK 888 at the top of transmembrane segment (TM) V and VI, either alone or as a group, did not affect the binding of these nonpeptide compounds. However, substitution of Ser290 in TM VII of the rat receptor with isoleucine present in the human receptor increased the affinity for FK 888 20-fold and that for CP 96345 6-fold, corresponding to an affinity that was only about 4-fold less than the affinity for the human NK-1 receptor. Full human-like affinity for FK 888 and CP 96,345 could be conveyed to the rat receptor by the combined substitution of Ser290 in TM VII to isoleucine and Leu116 in TM III to valine. The NK-2 receptor-selective compound SR 48,968 was found to bind with low affinity to the human NK-1 receptor but with 15-fold even lower affinity to the rat receptor. Substitution of residue 290, which is situated within the previously located binding site for this compound, could completely account for this difference. These data demonstrate that the species selectivities of the nonpeptide antagonists CP 96345, FK 888, and SR 48,968, independently of clear differences in their chemical structures and modes of discovery, have a similar structural basis, being dependent on two divergent residues that apparently are not involved in peptide agonist binding.
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PMID:The species selectivity of chemically distinct tachykinin nonpeptide antagonists is dependent on common divergent residues of the rat and human neurokinin-1 receptors. 750 41

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both alpha subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the alpha subunit with the delta or the gamma chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192-193, in three loops-forming binding domains (loops A-C). Other residues such as the negatively changed aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-alpha subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as alpha-, kappa-bungarotoxin and several alpha-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The alpha subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid-protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. (ABSTRACT TRUNCATED)
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PMID:Topology of ligand binding sites on the nicotinic acetylcholine receptor. 940 37

Neurotrophic keratopathy, which often follows damage to the trigeminal nerve, is clinically characterized by various types of epithelial disorders and melting of corneal stroma. To understand both the pathology of neurotrophic keratopathy and the physiological significance of corneal sensation, we investigated both the cellular and molecular functions of a sensory neurotransmitter, substance P, in corneal epithelial cells. Our findings prompted us to try a new mode of treatment for neurotrophic keratopathy. Substance P, a member of the tachykinin family, is an 11-amino-acid peptide. In an organ culture system using rabbit corneas, substance P alone had no effect on corneal epithelial migration. In the presence of insulin-like growth factor-1 (IGF-1), however, substance P synergistically facilitated corneal epithelial migration in proportion to the concentration of substance P or of IGF-1. Other neurotransmitters (acetylcholine, norepinephrine, serotonin etc.) or tachykinins (neurokinin A, eledoisin etc.) did not show this synergistic effect with IGF-1. Among receptors for the tachykinin family (NK-1, NK-2, or NK-3) only the NK-1 receptor system was involved in the synergistic effect of substance P and IGF-1 on corneal epithelial migration. IGF-1 affected neither the binding constant nor the number of sites of substance P receptors in corneal epithelial cells, suggesting that the synergistic effect was not regulated at the receptor level. Various extracellular signals activate the intracellular signal transduction system, thus amplifying specific biological functions. We found that the addition of inhibitors of protein kinase C or tyrosine kinase clearly inhibited the synergistic effect of substance P and IGF-1 on corneal epithelial migration, demonstrating that protein kinase C and tyrosine kinase are involved in the synergistic effect. During corneal epithelial wound healing, epithelial cells must attach to a provisional, extracellular fibronectin matrix. We previously reported that interleukin 6 and epidermal growth factor (EGF) facilitate corneal epithelial wound healing by activating the expression of fibronectin receptor (integrin). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that substance P and IGF-1 increased expression of mRNA for integrins alpha 5 and beta 1 in cultured corneal epithelial cells and also increased the number of cells that attached to a fibronectin matrix. These findings strongly suggest that substance P and IGF-1 synergistically increase corneal epithelial migration by activating the expression of integrin. Tachykinins share a five amino acid sequence, phenylalanine-free amino acid-glycine-leucine-methionine amide (FXGLM), at the C-terminus. Studying substance P, we found that a four amino acid sequence at the C-terminus, FGLM, was the minimum amino acid sequence for the synergistic effect on corneal epithelial migration. Structurally similar tetrapeptides mimicking other members of the tachykinin family isoleucine-glycine-leucine-methionine amide (IGLM), valine-glycine-leucine-methionine amide (VGLM), tyrosine-glycine-leucine-methionine amide (YGLM), and the tripeptide glycine-leucine-methionine amide (GLM) did not have any synergistic effect with IGF-1. Based on these findings in vitro, we investigated the effect of eye drops containing substance P plus IGF-1 or FGLM plus IGF-1 on the epithelial wound closure of rabbit corneas in vivo. Both combinations significantly facilitated corneal epithelial wound closure. In a clinical setting, the administration of substance P plus IGF-1 effectively treated corneal epithelial defects in a patient with Riley-Day syndrome, a disease in which corneal epithelial defects persist because of loss of corneal sensation and hypolacrimation. In a patient with neurotrophic keratopathy due to trigeminal nerve paralysis following surgery, eye drops containing FGLM plus IGF-1 eliminated superficial punctate staining. (ABSTRACT TRUNCATED)
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PMID:[Neurotrophic keratopathy--studies on substance P and the clinical significance of corneal sensation]. 943 58

Residues in transmembrane domain (TM)-III, TM-V, TM-VI, and TM-VII believed to be facing the deep part of the presumed main ligand-binding pocket of the NK1 receptor were probed by alanine substitution and introduction of residues with larger and/or chemically distinct side chains. Unaltered or even improved binding affinity for four peptide agonists, substance P, substance P-O-methyl ester, eledoisin, and neurokinin A, as well as normal EC50 values for substance P in stimulating phosphatidylinositol turnover indicated that these mutations did not alter the overall functional integrity of the receptor. The alanine substitutions in general had only minor effects on nonpeptide antagonist binding. However, the introduction of the larger and polar aspartic acid and histidine residues at positions corresponding to the monoamine binding aspartic acid in TM-III of the beta 2-adrenoceptor (ProIII:08, Pro112 in the NK1 receptor) and to the presumed monoamine interacting "two serines" in TM-V (ThrV:09, Thr201; and IleV:12, Ile204) impaired by > 100-fold the binding of a group of nonpeptide antagonists, including CP96,345, CP99,994, RP67,580, RPR100,893, and CAM4092. In contrast, another group of nonpeptide antagonists, LY303,870, FK888, and SR140,333, were little or not at all affected by the space-filling substitutions. Two of these compounds, FK888 and LY303,870, were those most seriously affected (75-89-fold) by alanine substitution of PheVI:20 located in the upper part of the main ligand-binding crevice. Surprisingly, substitution of AlaIII:11 (Ala115), which is located in the middle of TM-III, conceivably pointing toward TM-VII, with a larger valine residue increased the affinity for all 13 ligands tested, presumably by creating a closer interhelical packing. It is concluded that the introduction of larger side chains at positions at which molecular models indicate that this is structurally allowed can be a powerful method of locating ligand-binding sites due to the considerable difference between positive and negative results. Such steric hindrance mutagenesis strongly indicates that one population of nonpeptide antagonists bind in the deep pocket of the main ligand-binding crevice of the NK1 receptor, whereas another group of nonpeptide antagonists, especially SR140,333, was surprisingly resistant to mutational mapping in this pocket.
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PMID:Steric hindrance mutagenesis versus alanine scan in mapping of ligand binding sites in the tachykinin NK1 receptor. 944 45

The nicotinic acetylcholine receptor (AChR) is the paradigm of the neurotransmitter-gated ion channel superfamily. The pharmacological behavior of the AChR can be described as three basic processes that progress sequentially. First, the neurotransmitter acetylcholine (ACh) binds the receptor. Next, the intrinsically coupled ion channel opens upon ACh binding with subsequent ion flux activity. Finally, the AChR becomes desensitized, a process where the ion channel becomes closed in the prolonged presence of ACh. The existing equilibrium among these physiologically relevant processes can be perturbed by the pharmacological action of different drugs. In particular, non-competitive inhibitors (NCIs) inhibit the ion flux and enhance the desensitization rate of the AChR. The action of NCIs was studied using several drugs of exogenous origin. These include compounds such as chlorpromazine (CPZ), triphenylmethylphosphonium (TPMP+), the local anesthetics QX-222 and meproadifen, trifluoromethyl-iodophenyldiazirine (TID), phencyclidine (PCP), histrionicotoxin (HTX), quinacrine, and ethidium. In order to understand the mechanism by which NCIs exert their pharmacological properties several laboratories have studied the structural characteristics of their binding sites, including their respective locations on the receptor. One of the main objectives of this review is to discuss all available experimental evidence regarding the specific localization of the binding sites for exogenous NCIs. For example, it is known that the so-called luminal NCIs bind to a series of ring-forming amino acids in the ion channel. Particularly CPZ, TPMP+, QX-222, cembranoids, and PCP bind to the serine, the threonine, and the leucine ring, whereas TID and meproadifen bind to the valine and extracellular rings, respectively. On the other hand, quinacrine and ethidium, termed non-luminal NCIs, bind to sites outside the channel lumen. Specifically, quinacrine binds to a non-annular lipid domain located approximately 7 A from the lipid-water interface and ethidium binds to the vestibule of the AChR in a site located approximately 46 A away from the membrane surface and equidistant from both ACh binding sites. The non-annular lipid domain has been suggested to be located at the intermolecular interfaces of the five AChR subunits and/or at the interstices of the four (M1-M4) transmembrane domains. One of the most important concepts in neurochemistry is that receptor proteins can be modulated by endogenous substances other than their specific agonists. Among membrane-embedded receptors, the AChR is one of the best examples of this behavior. In this regard, the AChR is non-competitively modulated by diverse molecules such as lipids (fatty acids and steroids), the neuropeptide substance P, and the neurotransmitter 5-hydroxytryptamine (5-HT). It is important to take into account that the above mentioned modulation is produced through a direct binding of these endogenous molecules to the AChR. Since this is a physiologically relevant issue, it is useful to elucidate the structural components of the binding site for each endogenous NCI. In this regard, another important aim of this work is to review all available information related to the specific localization of the binding sites for endogenous NCIs. For example, it is known that both neurotransmitters substance P and 5-HT bind to the lumen of the ion channel. Particularly, the locus for substance P is found in the deltaM2 domain, whereas the binding site for 5-HT and related compounds is putatively located on both the serine and the threonine ring. Instead, fatty acid and steroid molecules bind to non-luminal sites. More specifically, fatty acids may bind to the belt surrounding the intramembranous perimeter of the AChR, namely the annular lipid domain, and/or to the high-affinity quinacrine site which is located at a non-annular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophi
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PMID:Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor. 974 59

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