Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors.

The family of mammalian tachykinin receptors consists of substance P receptor (SPR), neuromedin K receptor (NKR) and substance K receptor (SKR). In this investigation, tissue and regional distributions of the mRNAs for the three rat tachykinin receptors were investigated by blot-hybridization and RNase-protection analyses using the previously cloned receptor cDNAs. SPR mRNA is widely distributed in both the nervous system and peripheral tissues and is expressed abundantly in the hypothalamus and olfactory bulb, as well as in the urinary bladder, salivary glands and small and large intestines. In contrast, NKR mRNA is predominantly expressed in the nervous system, particularly in the cortex, hypothalamus and cerebellum, whereas SKR mRNA expression is restricted to the peripheral tissues, being abundant in the urinary bladder, large intestine, stomach and adrenal gland. Thus, the mRNAs for the three tachykinin receptors show distinct patterns of expression between the nervous system and peripheral tissues. Blot-hybridization analysis in combination with S1 nuclease protection and primer-extension analyses revealed that there are two large forms of SKR mRNA expressed commonly in the peripheral tissues, and two additional small forms of the mRNA expressed specifically in the adrenal gland and eye. These analyses also showed that the multiple forms of SKR mRNA differ in the lengths of the 5' mRNA portions, and that the two small forms of the mRNA, if translated, encode a truncated SKR polypeptide lacking the first two transmembrane domains. This investigation thus provides the comprehensive analysis of the distribution and mode of expression of the mRNAs for the multiple peptide receptors and offers a new basis on which to interpret the diverse functions of multiple tachykinin peptides in the CNS and peripheral tissues.     

The primary structure and gene organization of human substance P and neuromedin K receptors.

The gene organization and amino acid sequences of human substance P and neuromedin K receptors (SPR and NKR, respectively) are reported on the basis of molecular cloning and sequence determination of genomic DNA containing the respective receptor gene. The human SPR and NKR genes, unlike many other genes for G-protein-coupled receptors, (G protein, guanyl-nucleotide-binding-regulatory protein), contain introns which interrupt the protein-coding regions into 5 exons. The human SPR and NKR genes extend over 60 kb and 45 kb, respectively and are considerably larger than the human substance K receptor (SKR) gene consisting of 12 kb. All 4 introns, however, are located at equivalent positions of the three tachykinin receptor genes, suggesting that they evolved from a common ancestral gene. Human SPR and NKR consist of 407 and 465 amino acid residues, respectively, each possessing structural features characteristic of the members of G-protein-coupled receptors. The human and rat receptors show a common tendency of distinctly segmented sequence conservation and divergence among the three receptors, and the observed sequence conservation and divergence would contribute to the emergence of similar but distinct properties of the three receptors. Furthermore, the amino acid sequences and the gene sizes are more closely related between SPR and NKR than between SKR and NKR, suggesting that the SPR gene evolved from the primordial NKR gene after a gene duplication to form the NKR and SKR genes.     

Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected Chinese hamster ovary cells.

The mammalian tachykinin system consists of three distinct peptides, substance P, substance K, and neuromedin K, and possesses three corresponding receptors. In this investigation we examined intracellular signal transduction of the individual tachykinin receptors by transfection and stable expression of these receptor cDNAs in Chinese hamster ovary cells. The three receptors commonly showed a rapid and marked stimulation in both phosphatidylinositol (PI) hydrolysis and cyclic AMP formation in response to tachykinin interaction. Direct linkage of the three receptors to both phospholipase C and adenylate cyclase was evidenced by the finding that tachykinin, added together with GTP, activated these enzyme activities in membrane preparations derived from tachykinin receptor-expressing cells. The stimulation of cyclic AMP formation was less efficient than that of PI hydrolysis in receptor-expressing cells as well as their membrane preparations (about 1 order of magnitude difference in the effective peptide concentrations). However, the stimulatory responses of the PI hydrolysis and cyclic AMP formation in both receptor-expressing cells and their membrane preparations were induced in complete agreement with the tachykinin binding selectivity of each subtype of the receptors. This investigation demonstrated unequivocally that the tachykinin receptors have the potential to couple directly to both phospholipase C and adenylate cyclase and to stimulate PI hydrolysis and cyclic AMP formation.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.     

Molecular cloning, structural characterization and functional expression of the human substance P receptor.

A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-1-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP much greater than neurokinin A greater than neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver.     

Functional divergence of glycoprotein hormone receptors

Two lamprey glycoprotein hormone receptors (lGpH-R I and II) highly similar with gnathostome GpH-Rs were cloned from sea lamprey testes and thyroid, respectively. Vertebrate glycoprotein protein receptors have a large extracellular domain (ED) containing a leu rich domain (LRD) linked to a rhodopsin-like transmembrane domain (TMD) through a highly divergent linker region (signal specificity domain, SSD or 'hinge' region) and a third major segment, the intracellular domain. To determine the potential roles of the different domains in the activation of the receptor following ligand-receptor binding, functional assays were performed on lGpH-R I/rat luteinizing hormone (LH)-R domain swapped chimeric receptors. These results show that the functional roles of the lamprey glycoprotein-receptor I (lGpH-R I) domains are conserved compared with its Gnathostome homologs. The ability of different glycoprotein hormones to activate chimeric lamprey/rat receptors suggests that the selectivity of the GpH-Rs in respect to their ligands is not controlled exclusively by a single domain but is the result of specific interactions between domains. We hypothesize that these interactions were refined during millions of years of co-evolution of the receptors with their cognate ligands under particular intramolecular, intermolecular and physiological constraints.     

Cloning and expression of the human substance K receptor and analysis of its role in mitogenesis.

The primary structure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfected NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I-labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substance K and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfected cells led to a rapid increase in the production of total inositol phosphates and the release of Ca2+ from internal stores. Growth of the cells transfected with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth factor receptor when expressed in mouse 3T3 cells.     

The polyprotein nature of substance P precursors

Substance P and related tachykinin peptides probably act as neurotransmitters or modulators of neurotransmission, and regulate biological processes as diverse as salivary secretion and transmission of pain signals. Substance P peptide sequences are expressed in three distinct mRNAs that are generated from one gene by differential RNA splicing. In addition to substance P, as many as three other tachykinin peptides can be generated from the polyprotein precursors by differential posttranslational processing. Three tachykinin receptor subtypes have been extensively characterized which differentially interact with the naturally occurring tachykinin peptides. Therefore, the generation of diversity of tachykinin peptides results from differential precursor RNA splicing and differential posttranslational processing. The specificity of peptide responses is the result of selective receptor subtype expression.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

Effects of activators and antagonists of the neuropeptides substance P and substance K on cell proliferation in planarians

Substance P and substance K (Neurokinin A) are mammalian peptides belonging to the tachykinin family. Both have been studied extensively, are widely distributed in both central and peripheral mammalian nervous systems, and seem to be involved in pain reactions and inflammatory responses. We report here that substance P and substance K, as well as Epidermal Growth Factor (EGF), are potent mitogens, at micro and nanomolar concentrations, for planarian cells. This stimulation is inhibited by the substance P and substance K antagonist spantide, while capsaicin, a pungent agent of capsicum peppers that destroys sensory neurons, stimulates cell division, probably through release of substance P. These results, jointly with the reported stimulation of cell division by naloxone and its inhibition by Met-Enkephalin (Bagu?a, 1986), both probably acting on tachykinin release, suggest that target cells, the neoblasts, must have in their cell membranes numerous receptors for growth hormones and neuropeptides analogous to their mammalian counterparts.     

Changes in amino-terminal portion of human B2 receptor selectively increase efficacy of synthetic ligand HOE 140 but not of cognate ligand bradykinin

Recently, we have shown that a widely used antagonist of the human bradykinin B(2) receptor (B(2)R) HOE 140 acts as a full agonist of the chicken ornithokinin receptor (B(o)R). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B(2)R were substituted with the corresponding segments of B(o)R. Ligand efficacy of synthetic ligand HOE 140 decreased in the order B(o)R > CKR-2 > CKR-1 > B(2)R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R*, thereby increasing the ratio of R* over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species.     

Role of transmembrane helix IV in G-protein specificity of the angiotensin II type 1 receptor

G-protein activation by G-protein coupled receptors (GPCRs) is accomplished through proper interaction with the cytoplasmic loops rather than through sequence-specific interactions. However, the mechanism by which a specific G-protein is selected by a GPCR is not known. In the current model of GPCR activation, agonist binding modulates helix-helix interactions, which is necessary for fully determining G-protein specificity and stimulation of GDP/GTP exchange. In this study, we report that a single-residue deletion in transmembrane helix IV leads the angiotensin II type 1 (AT(1)) receptor chimera CR17 to retain GTP-sensitive high affinity for the agonist angiotensin II but results in complete inactivation of intracellular inositol phosphate production. The agonist dissociation profile of CR17 in the presence of guanosine 5'-3-O-(thio)triphosphate suggests that the activation-induced conformational changes of the chimeric receptor itself remain intact. Insertion of an alanine at position 149 (CR17triangle down149A) in this chimera rescued the inactive phenotype, restoring intracellular inositol phosphate production by the chimera. This finding suggests that in the wild-type AT(1) receptor the orientation of transmembrane helix IV-residues following Cys(149) is a key determinant for effectively distinguishing among various structurally similar G-proteins. The results emphasize that the contacts within the membrane-embedded portion of transmembrane helix IV in the AT(1) receptor is important for specific G-protein selection.     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Localization of agonist and antagonist binding domains of the human neurokinin-1 receptor.

To identify the molecular determinants of ligand-receptor interactions, the extracellular domain of the human neurokinin-1 receptor was systematically substituted with the corresponding sequences from the other two neurokinin receptor subtypes. Three residues within the first extracellular segment and 2 residues of the second segment are required for the optimal binding of all three natural peptide agonists. The divergent nature of 4 of the 5 residues supports the hypothesis that the peptide binding site on the neurokinin-1 receptor is not highly conserved in the other two receptor subtypes. In contrast, substitution of part of the third extracellular segment and the fourth extracellular segment with the corresponding amino acids of the human neurokinin-3 receptor results in an increase in neurokinin B affinity without affecting substance P binding, suggesting that the two peptides do not interact with the same set of functional groups on the receptor. Among the four extracellular regions, only parts of the third and fourth segments affect the binding of the quinuclidine antagonist L-703,606, and these two regions may partially account for the neurokinin-1 receptor subtype specificity of this non-peptide antagonist. These studies demonstrate that both the extracellular and transmembrane domains of the neurokinin-1 receptor are involved in the binding of substance P and related peptides.     

Molecular characterization of rat substance K receptor and its mRNAs

The nucleotide sequence and the amino acid sequence for rat substance K receptor were deduced by molecular cloning and sequence analysis of its cDNAs. The rat substance K receptor consists of 390 amino acid residues and belongs to the family of G protein-coupled receptors. The comparison of the amino acid sequences of the rat and bovine substance K receptors indicated that they are highly homologous in the regions covering seven putative transmembrane domains, and this similarity is particularly remarkable in the transmembrane segments III and VII and their surrounding regions. RNA blot hybridization analysis showed that the rat substance K receptor is encoded by two species of mRNAs which differ in the lengths of the extreme 5' sequence of the 5'-untranslated regions. This analysis also indicated that the substance K receptor mRNAs are expressed in the gastrointestinal tract. Interestingly, no appreciable substance K receptor mRNAs were detected in poly(A)+ RNAs isolated from the brain and spinal cord, even though these tissues are known to not only contain substance K but also express the mRNA encoding the substance K precursor.     

The extracellular domain of the neurokinin-1 receptor is required for high-affinity binding of peptides.

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.     

Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons

Rat genomic clones were used to quantitate preprotachykinin mRNAs in the rat basal ganglia, while the tachykinin peptide products substance P and substance K were measured by radioimmunoassay. Administration of the dopamine antagonist (antipsychotic) drug haloperidol significantly decreased substance P, substance K, and both alpha (substance P encoding) and beta (substance P/substance K encoding) preprotachykinin mRNAs, suggesting a drug-induced decrease in striatonigral tachykinin biosynthesis. The time course for decreased preprotachykinin mRNAs and tachykinins apparently parallels the period of maximum risk for the development of certain antipsychotic drug-induced extrapyramidal side effects seen clinically. Tachykinin interaction with dopamine neurons may play an important role in the modulation of basal ganglia function.     

Tachykinin regulation of cholinergic transmission in the limbic/prefrontal territory of the rat dorsal striatum: implication of new neurokinine 1-sensitive receptor binding site and interaction with enkephalin/mu opioid receptor transmission

The tachykinin neurokinin 1 receptors (NK₁Rs) regulation of acetylcholine release and its interaction with the enkephalin/mu opioid receptors (MORs) transmission was investigated in the limbic/prefrontal (PF) territory of the dorsal striatum. Using double immunohistochemistry, we first showed that in this territory, cholinergic interneurons contain tachykinin NK₁Rs and co-express MORs in the last part of the light period (afternoon). In slices of the striatal limbic/PF territory, following suppression of the dopaminergic inhibitory control of acetylcholine release, application of the tachykinin NK₁R antagonist, SSR240600, markedly reduced the NMDA-induced acetylcholine release in the morning but not in the afternoon when the enkephalin/MOR regulation is operational. In the afternoon, the NK₁R antagonist response required the suppression of the enkephalin/MOR inhibitory control of acetylcholine release by βfunaltrexamine. The pharmacological profile of the tachykinin NK₁R regulation tested by application of the receptor agonists [[Pro⁹]substance P, neurokinin A, neuropeptide K, and substance P(6–11)] and antagonists (SSR240600, GR205171, GR82334, and RP67580) indicated that the subtype of tachykinin NK₁R implicated are the new NK₁-sensitive receptor binding site. Therefore, in the limbic/PF territory of the dorsal striatum, endogenous tachykinin facilitates acetylcholine release via a tachykinin NK₁R subtype. In the afternoon, the tachykinin/NK₁R and the enkephalin/MOR transmissions interact to control cholinergic transmission.