Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.     

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.     

Further definition of the substance P (SP)/neurokinin-1 receptor complex. MET-174 is the site of photoinsertion p-benzoylphenylalanine4 SP

The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.     

Tachykinins stimulate a subset of mouse taste cells

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.     

Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin

To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the response to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-1-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist and antagonist binding to tachykinin peptide NK-2 receptors

The binding of tachykinin peptides and fragments to NK-2 receptor sites in hamster urinary bladder membranes was examined and compared to binding to NK-1 receptor sites in rat submandibular gland. Neurokinin A (NKA) and its C-terminal fragments bound with highest NK-2 affinity and selectivity. N-terminal fragments of NKA did not bind to either type of receptor. Kassinin and eledoisin were NK-2 selective while physalaemin, phyllomedusin, and uperolein were NK-1 selective. Of fifteen tachykinin antagonists examined, none exhibited appreciable affinity or selectivity (relative to agonists) for NK-1, NK-2, or rat cerebral cortical NK-3 receptor sites. NKA binding to NK-2 sites was stimulated by Mn++ greater than Mg++ greater than Ca++. At the optimal concentration, the Mn++ stimulation was due to both an increased Bmax and increased affinity. The nonhydrolyzable guanine nucleotide, GppNHp, reduced agonist binding but not antagonist binding to NK-2 receptor sites. The nucleotide effect was due to a reduction in both Bmax and affinity and was potentiated by Mn++. The results indicate that tachykinin NK-2 receptor sites possess distinct structural requirements for agonists and are linked to a G-protein coupling system.     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

A potent and selective agonist for NK-2 tachykinin receptor

Replacement of the glycine in position 8 of the C-terminal heptapeptide NKA(4-10) with beta-alanine give rise to a potent and selective agonist for the NK-2 tachykinin receptor. The affinity of [beta-Ala8]-NKA(4-10) to the NK-2 receptor is enhanced by almost one order of magnitude as compared to NKA(4-10), while affinity decreases at about the same extent at NK-1 and NK-3 receptors, respectively. Synthesis and biological activities of a series of NKA(4-10) analogues systematically replaced in each position with beta-alanine are also reported.     

Met174 side chain is the site of photoinsertion of a substance P competitive peptide antagonist photoreactive in position 8

Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8.     

Selective agonists for receptors of substance P and related neurokinins

Neurokinins and their receptors are a complex system consisting of at least three endogenous agents--substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)--and their corresponding receptor types, respectively, NK-1, NK-2, and NK-3. Investigations on receptors have been made using sensitive and fairly selective pharmacological preparations (the dog carotid artery for the NK-1, the rabbit pulmonary artery devoid of endothelium for the NK-2, and the rat portal vein for the NK-3 receptor), and some natural peptides of mammalian and nonmammalian origin. Because of the nonselectivity of the natural peptides, analogues of the neurokinins have been found that act on one receptor only and show therefore high selectivity. The selective agonists [Sar9,Met(O2)11]SP, [Nle10]NKA (4-10), and [MePhe7]-NKB have been used successfully for (a) characterizing the three neurokinin receptors, (b) identifying isolated organs whose responses to neurokinins depend on the activation of a single (monoreceptor systems) or of more than one (multireceptor systems) receptor, and (c) elucidating some of the physiological function of the three receptor types. It is suggested that NK-1 mediate peripheral vasodilatation and exocrine secretions, NK-2 stimulate bronchial muscles and facilitate the release of catecholamines, and NK-3 promote the release of acetylcholine in peripheral organs.     

A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.     

Apparent molecular weight of substance P/neurokinin-1 receptors determined using a photoaffinity labelled probe, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P

A photoreactive derivative, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P (SP) was prepared and iodinated using carrier-free [125I] to determine the apparent molecular weight of one sub-type of neurokinin (NK) receptor, the SP/NK-1 type. The unlabelled analogue competed for [3H]-SP sites with an IC50 of 10 nM. The radioactive photoprobe (KD approximately 0.17 nM, Bmax = 15.6 fmol/mg protein) was used to photoaffinity label membranes prepared from rat brain. Autoradiographs revealed that a single band with an apparent molecular weight of 46,000 daltons was specifically labelled. This labelling was inhibited by non-radioactive SP in a concentration-dependent manner (1.0 nM-0.1 mM) suggesting that the observed labelling represents the SP/NK-1 receptor type.     

Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.     

Cgamma H2 of Met174 side chain is the site of covalent attachment of a substance P analog photoactivable in position 5

Analogs of substance P (H-RPKPQQFFGLM-NH(2)) incorporating a photoreactive para-benzoyl-l-phenylalanine (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence have been synthesized and pharmacologically characterized previously as full NK-1 receptor agonists. In this study we show that all analogs, [BAPA(0), (p-Bzl)Phe(x), Met(O(2))(11)]SP also display high yields (40-70%) of NK-1 receptor photolabeling. To identify the site of photoinsertion in the receptor, covalent ligand/receptor complexes were digested with enzymes or chemically cleaved with cyanogen bromide and purified with streptavidin-coated magnetic beads before matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Only the analog photoreactive at position 5 gave irreversible, reproducible, and unequivocal covalent linkage. Sequential digestions of the covalent complex, substance P analog photoreactive at position 5/NK-1 receptor, with trypsin, endo-GluC and carboxypeptidase Y, led to the identification of the tripeptide (173)TMP(175) in the second extracellular loop of the hNK-1 receptor as the site of photoinsertion. Reaction of cyanogen bromide on the pentapeptide TMPSR did not yield the expected cleavage on the carboxylic side of methionine. The high precision of mass spectrometry analysis on the mass measured led us to determine that C(gamma)H(2) of Met(174) was the site of covalent linkage of the photoreactive substance P analog. Such an insertion (photolinked ligand) on its C(gamma)H(2) renders methionine refractory to CNBr cleavage.     

Synthesis and biological activity of NK-2 selective tachykinin antagonists containing D-tryptophan

By introducing D-Trp in position 6 and 8 along with pyroglutamic acid (Pyr) in position 4 or Nle in position 10 of NKA(4-10) we have obtained selective although weak NK-2 tachykinin receptor antagonists. Similar substitutions, previously reported on the sequence of SP, gave rise to nonselective antagonists presumably for the limited selectivity of the agonist used as template. Further modifications like the addition of a third D-Trp in position 9 gave rise to more potent but less selective antagonists, thus showing that each amino acid substitution can dramatically affect selectivity.     

SR142801 behaves as a tachykinin NK-3 receptor agonist on a spinal nociceptive reflex in the rat

Effects of two commonly used tachykinin NK-3 receptor antagonists (SR 142801 and R820) intrathecally (i.t.) administered were assessed in the rat tail-flick test. SR142801 and its (R)-enantiomer SR142806 (1.3, 6.5 and 65 nmol) were found as potent as senktide and [MePhe7]NKB (NK-3 selective agonists) to induce transient antinociceptive effects. Naloxone (10 microg) and R820 (6.5 nmol) blocked reversibly the responses to 6.5 nmol senktide, [MePhe7]NKB, SR142801 and SR142806 when administered i.t. 15 min earlier. However, the antinociceptive responses induced by SR142801 and SR142806 were not affected by i.t. pretreatments with NK-1 (6.5 nmol SR140333) and NK-2 (6.5 nmol SR48968) receptor antagonists. In control experiments, the NK-1 and NK-2 antagonists prevented the hyperalgesic effects to NK-1 ([Sar9,Met(O2)11]SP) and NK-2 ([beta-Ala8] NKA(4-10)) receptor agonists (6.5 nmol i.t.), respectively. R820 had no direct effect on nociceptive threshold and failed to alter angiotensin II-induced antinociception. The data suggest that the antinociceptive effect of SR142801 is due to an agonist effect at NK-3 receptor in the rat spinal cord that involves a local opioid mechanism. These results can be best explained by the existence of inter-species NK-3 receptor subtypes.     

Cutting edge: hemokinin has substance P-like function and expression in inflammation

Substance P (SP) belongs to the tachykinin family of molecules. SP, cleaved from preprotachykinin A, is a neuropeptide and a proinflammatory leukocyte product. SP engages neurokinin 1 receptor (NK-1R) to stimulate cells. Hemokinin (HK) is another tachykinin that binds NK-1R. HK comes from preprotachykinin C, which is distinct from preprotachykinin A. We determined whether HK functions like SP at inflammatory sites. Preprotachykinin C mRNA was in murine schistosome granulomas and intestinal lamina propria mononuclear cells. Granuloma T cells and macrophages expressed preprotachykinin C mRNA. HK bound granuloma T cell NK-1R with high affinity. SP and HK stimulated IFN-gamma production with equal potency. NK-1R antagonist blocked the effect of SP and HK on IFN-gamma secretion. Thus, both HK and SP are expressed at sites of chronic inflammation and share cell origin, receptor, and immunoregulatory function. Two distinct but functionally overlapping tachykinins govern inflammation through NK-1R at sites of chronic inflammation.     

The substance P/neurokinin-1 receptor system in lung cancer: Focus on the antitumor action of neurokinin-1 receptor antagonists

The last decades have seen no significant progress in extending the survival of lung cancer patients and there is an urgent need to improve current therapies. The substance P (SP)/neurokinin-1 receptor (NK-1R) system plays an important role in the development of cancer: SP and NK-1R antagonists respectively induce cell proliferation and inhibition in human cancer cell lines. No study of the involvement of this system in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells has been carried out in depth. Here, we demonstrate the involvement of the SP/NK-1R system in human H-69 (SCLC) and COR-L23 (NSCLC) cell lines: (1) they express isoforms of the NK-1R and mRNA for the NK-1R; (2) they overexpress the tachykinin 1 gene; (3) the NK-1R is involved in their viability; (4) SP induces their proliferation; (5) NK-1R antagonists (Aprepitant (Emend), L-733,060, L-732,138) inhibit the growth of both cell lines in a concentration-dependent manner; (6) the specific antitumor action of these antagonists against such cells occurs through the NK-1R; and (7) lung cancer cell death is due to apoptosis. We also demonstrate the presence of NK-1Rs and SP in all the human SCLC and NSCLC samples studied. Our findings indicate that the NK-1R may be a promising new target in the treatment of lung cancer and that NK-1R antagonists could be new candidate antitumor drugs in the treatment of SCLC and NSCLC.