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.     

Identification of a segment of the gene for the substance B receptor in the human DNA genome using the polymerase chain reaction]

Polymerase chain reaction was applied to human genomic DNA using primers corresponding to the rat substance P receptor cDNA. As a result, a fragment of 94 b.p. was isolated identical to the fragment 771-864 of the above-mentioned cDNA, with the exception of the G796----A substitution (Val----Ile in the amino acid sequence). A comparison of the established sequence with the published structures of tachykinin receptors of NK-1, NK-2 and NK-3 types allows its assignment to the substance P receptor (NK-1 tachykinin receptor) gene detected in the human genome.     

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.     

Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors

We screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to conserved amino acid sequences from the five known rat somatostatin receptors. This yielded alignment with a Drosophila genomic clone that contained a DNA sequence coding for a protein, having amino acid sequence identities with the rat galanin receptors. Using PCR with Drosophila cDNA as a template, and oligonucleotide probes coding for the exons of the presumed Drosophila gene, we were able to clone the cDNA for this receptor. The Drosophila receptor has most amino acid sequence identity with the three mammalian galanin receptors (37% identity with the rat galanin receptor type-1, 32% identity with type-2, and 29% identity with type-3). Less sequence identity exists with the mammalian opioid/nociceptin-orphanin FQ receptors (26% identity with the rat micro opioid receptor), and mammalian somatostatin receptors (25% identity with the rat somatostatin receptor type-2). The novel Drosophila receptor gene contains ten introns and eleven exons and is located at the distal end of the X chromosome.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

Cloning and expression of a rat neuromedin K receptor cDNA

Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K receptor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins: neuromedin K greater than substance K greater than substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are though to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.     

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.     

Characterization and comparative structural features of the gene for human interstitial retinol-binding protein

We have cloned the gene for human interstitial retinol-binding protein (IRBP) and compared its nucleotide sequence with that of the corresponding cloned cDNA. The human IRBP gene is approximately 9.5 kilobase pairs (kbp) in length and consists of four exons separated by three introns. The introns are 1.6-1.9 kbp long. The gene is transcribed by photoreceptor and retinoblastoma cells into an approximately 4.3-kilobase mRNA that is translated and processed into a glycosylated protein of 135,000 Da. The amino acid sequence of human IRBP can be divided into four contiguous homology domains with 33-38% identity, suggesting a series of gene duplication events. In the gene, the boundaries of these domains are not defined by exon-intron junctions, as might have been expected. The first three homology domains and part of the fourth are all encoded by the first large exon, which is 3,180 base pairs long. The remainder of the fourth domain is encoded in the last three exons, which are 191, 143, and approximately 740 base pairs long, respectively. This unusual structure is shared with the bovine IRBP gene. A large (1.7 kbp) fragment appears to have been lost from the 3'-noncoding region of the last human exon. We conclude that the human and bovine genes have similar evolutionary histories.     

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.     

Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones.

The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5' and 3' ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds 125I-BHSP with a Kd of 0.35 +/- 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.     

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.     

Structural Organization of the Mouse and Human GALR1 Galanin Receptor Genes (GalnrandGALNR) and Chromosomal Localization of the Mouse Gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15–20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouseGalnrgene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the humanGALNRgene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate

The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandb?ck, D., Rodén, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.