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.     

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.     

Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors.

The mammalian tachykinin receptors belong to the family of G protein-coupled receptors and consist of the substance P, substance K and neuromedin K receptors (SPR, SKR and NKR). We constructed 14 chimeric receptors in which seven transmembrane segments were sequentially exchanged between the rat SPR and SKR and examined the subtype specificity of the chimeric receptors by radioligand binding and inositol phosphate measurements after transfection into COS cells. All chimeric receptors showed maximum responses in agonist-induced inositol phosphate stimulation. Detailed analysis of five receptors with agonist selectivity similar to SPR indicated that the selectivity is mainly determined by the region extending from transmembrane segment II to the second extracellular loop together with a minor contribution of the extracellular N-terminal portion. This conclusion was more directly confirmed by an additional chimeric formation in which the introduction of the above middle portion of SPR into the corresponding region of SKR conferred a high affinity binding to substance P. The tachykinin receptors can thus be divided into two functional domains: the region covering transmembrane segments V-VII and responsible for fundamental recognition of the common tachykinin sequence; and its preceding portion involved in evoking subtype specificity by interacting with the divergent sequences of the peptides.     

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.     

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.     

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland.

A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary cDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus oocytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (¹²⁵I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

Demonstration of two distinct tachykinin receptors in rat brain cortex

Eledoisin and substance P are members of a class of peptides termed tachykinins. They share a similar spectrum of biological activities but their relative potencies in various pharmacological assays differ. We have investigated whether there is more than one receptor for these tachykinins in rat brain cortex membranes. 125I-Bolton Hunter-conjugated eledoisin specifically binds to rat brain cortex membranes with high affinity. The binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). Scatchard analysis of the binding of this ligand is curvilinear suggesting that there are two binding sites with KD values of 0.9 +/- 0.7 nM and 20 +/- 10 nM. We tested various analogs and fragments of substance P and eledoisin for their ability to inhibit the binding of 125I-Bolton Hunter-conjugated eledoisin and 125I-Bolton Hunter-conjugated substance P to these membranes. The following peptides are more potent as inhibitors of the 125I-Bolton Hunter-conjugated eledoisin binding site than of the 125I-Bolton Hunter-conjugated substance P binding site: nonradioactive Bolton Hunter-conjugated eledoisin (greater than 100-fold), eledoisin (12-fold), kassinin (22-fold), neuromedin K (greater than 58-fold), and pyroglutamyl substance P(6-11)hexapeptide (4-fold). In contrast, substance P (21-fold), physalaemin (8-fold), and substance P methyl ester (1200-fold) were more potent as inhibitors of 125I-Bolton Hunter-conjugated substance P binding. These results suggest that these two ligands may bind to distinct receptors. 125I-Bolton Hunter-conjugated substance P binds specifically to rat parotid cell receptors, but 125I-Bolton Hunter-conjugated eledoisin does not, indicating that parotid cells contain only one of the receptor subtypes. The cortex membrane binding of both ligands is stimulated by low concentrations of MnCl2 (ED50 = 0.05 mM) and is inhibited by guanylyl-5'-(beta, gamma-imido)diphosphate (IC50 = 0.5 microM).     

A cDNA presumptively coding for the alpha subunit of the receptor with high affinity for immunoglobulin E

Rat mast cells and a neoplastic analogue such as rat basophilic leukemia (RBL) cells have receptors that have exceptionally high affinity for immunoglobulin E (IgE). When aggregated, these receptors induce cellular degranulation. The alpha chain of the receptor contains the binding site for IgE; the function(s) of the noncovalently associated beta and gamma chains is (are) still undefined. Using a cDNA library constructed from the mRNA of RBL cells, we have isolated a cDNA clone whose sequence predicts a putative 23-residue signal peptide, followed by a sequence that accurately predicts the amino acid composition, the peptide molecular weight, and six peptide sequences (encompassing 59 residues or 26% of the total number) determined for the alpha chain by direct analysis. These findings provide strong evidence that the cDNA codes for the alpha chain, even though expression has not been unambiguously achieved. The sequence suggests that the alpha chain contains a 180-residue extracellular portion with two homologous domains of approximately 35 residues, a 20-residue transmembrane segment containing an aspartic acid, and a 27-residue cytoplasmic portion containing 9 basic amino acids. The sequence shows no homology with the low-affinity receptor for IgE from lymphocytes but over 30% homology with an Fc gamma receptor.     

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.     

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.     

Cloning of two additional catecholamine receptors from rat brain

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.     

Structure and functional expression of the cloned rat neurotensin receptor.

A functional cDNA clone for the rat neurotensin receptor was isolated by combining molecular cloning in an RNA expression vector with an electrophysiological assay in Xenopus oocytes. The neurotensin receptor consists of 424 amino acids with seven putative transmembrane domains and belongs to the family of G protein-coupled receptors. The cloned receptor expressed in mammalian cells or in Xenopus oocytes shows a selective and high-affinity binding to neurotensin peptides and undergoes potent desensitization by repeated application of neurotensin. The neurotensin receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. This investigation discloses the molecular nature of the neurotensin receptor, which mediates the diverse neuronal and peripheral actions of neurotensin by effecting the G protein-associated second messenger system.     

Mammalian tachykinin-induced hydrolysis of inositol phospholipids in rat brain slices

The mammalian tachykinins substance K, neuromedin K and substance P stimulated inositol phospholipid hydrolysis in paired coronal sections through the rat brain. In contrast, none of these peptides had any effect on either basal or forskolin-stimulated cyclic AMP levels. The present results therefore implicate inositol phospholipid hydrolysis as a possible second messenger system mediating the effects of substance K and neuromedin K in addition to substance P.     

Similar Rates of Phosphatidylinositol Hydrolysis Following Activation of Wild-Type and Truncated Rat Neurokinin-1 Receptors

Abstract: The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC₅₀ of substance P-induced accumulation of ³H-inositol phosphates. Substance P-induced responses continued for 30–45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of ³H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.