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.     

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.     

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.     

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.     

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.     

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.     

Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family.

We report the complete sequence of the gene encoding mouse glial fibrillary acidic protein (GFAP), the intermediate filament (IF) protein specific to astrocytes. The 9.8 kb gene includes nine exons separated by introns ranging in size from 0.2 to 2.5 kb. A comparison of the organization of the GFAP gene with that of genes encoding other IF proteins reveals that the structure of IF genes is highly conserved in spite of considerable divergence at the amino acid level. Thus, most of the evolutionary events leading to the placement of introns in IF genes must have occurred prior to the duplication and subsequent divergence of IF genes from a presumptive common ancestral sequence. The conserved gene organization is unrelated to structural features of IF proteins. A curious feature of the GFAP gene is the large number of repeated sequences found in the introns. Six tracts of reiterated di- or trinucleotides are present, plus tandem repeats of two different novel sequences. One repeat is unique to the GFAP gene; the other occurs elsewhere in the mouse genome, although at relatively low frequency.     

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.     

Molecular characterization of a novel glycoprotein hormone G-protein-coupled receptor

We report the molecular characterization of a novel G-protein-coupled receptor, GPR48, that resembles proteins in the glycoprotein hormone receptor family. The full-length human GPR48 cDNA is comprised of 951 amino acids. The large extracellular amino terminus of 538 residues is composed of seventeen leucine-rich repeats (LRR). The genomic structure of GPR48 has several features in common with genes in the glycoprotein hormone receptor family. Analogous to these receptors, most of the LRR are encoded on single small exons, and the last exon encodes the seven transmembrane segments. The complete gene spans more than 60 kb with 18 exons and 17 introns. Northern blot analysis demonstrated high expression of GPR48 in the adult human pancreas, with moderate levels of expression in placenta, kidney, brain, and heart. Additionally, this receptor is expressed as early as 7 days post coitus in the mouse, indicating its potential involvement in development.     

Tandem linkage of human CSF-1 receptor (c-fms) and PDGF receptor genes

A 5' untranslated exon of the human CSF-1 receptor gene (c-fms) is separated by a 26 kb intron from the 32 kb receptor coding sequences. Nucleotide sequence analysis of cloned genomic DNA revealed that the 3' end of the PDGF receptor gene is located less than 0.5 kb upstream from this exon. Similarities in chromosomal localization, organization, and encoded amino acid sequences suggest that the genes encoding the CSF-1 and PDGF receptors arose through duplication. The as yet unidentified c-fms promoter/enhancer sequences may be confined to the nucleotides separating the two genes or could potentially lie within the PDGF receptor gene itself.     

A Novel Human Gene Encoding a G-Protein-Coupled Receptor (GPR15) Is Located on Chromosome 3

We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have namedGPR15.A comparison of the amino acid sequence of the receptor encoded byGPR15with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1.GPR15was mapped to human chromosome 3q11.2–q13.1.     

Comparative structure and evolution of murine CR2. The homolog of the human C3d/EBV receptor (CD21)

The complete nucleotide sequence of murine complement receptor type 2 (CR2) was determined from two overlapping cDNA clones derived from a lambda gt11 library of late pre-B cell origin. Comparison of the predicted sequence of the 1014 amino acid murine homolog with that of human CR2 revealed marked evolutionary conservation. The murine molecule was 65% identical to human CR2 overall, lacking a single repetitive sequence variably present in man. The 15 approximately 60-75 amino acid short consensus repeats (SCR) that constitute the entire extracellular domain of murine CR2 were 53 to 81% identical to and could be directly aligned with the human protein. As reported, the cytoplasmic tail shared 79% amino acid identity with human CR2, whereas that of the transmembrane was only 33%. Murine CR2 contained 16 potential N-linked glycosylation sites of which 6 were conserved, 4 altered, and 6 lost during human evolution. The hydropathicity profile of the two molecules was nearly colinear with some variation in the N-terminal region of the first repeat, as well as within the sixth and twelfth repeats. RNA blot analysis revealed a approximately 4.0 to 5.0 kb message in murine B lymphocytes, which was absent in T lymphocytes (thymus and spleen), liver, brain, lung, kidney, and heart. A method was devised to more precisely compare the repeat structures. An identity matrix analysis suggests that human ancestral CR2 evolved before divergence of the rodent and primate branches of the evolutionary tree through a series of predictable gene duplications, possibly giving rise to the precursor of human CR1 and murine CRY. The marked structural similarity between the human and murine receptors suggests functional conservation as well.     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.     

Comparative analysis of the human type 1a and bovine type 1 papillomavirus genomes.

The DNA sequences of the genomes of the bovine type 1 and human type 1a papillomaviruses were compared. The overall organization of both genomes is very similar. Three areas of maximal homology were found in the L1 and E1/E2 genes, and at the beginning of L2. The conservation of homologous amino acid sequences encoded in the open reading frames argues that these segments represent real genes or exons. Within these segments, however, only certain domains of the putative proteins are preferentially conserved. Two polypeptide chains show homologous arrangement of the cysteine residue clusters Cys-X-X-Cys, despite a lack of conservation of the rest of the amino acid sequence. A significant sequence divergence in a region where the three reading frames are open suggests that papillomavirus genomes have evolved not solely by accumulation of point mutations. Conserved sequences were also found in the noncoding region, and their possible involvement in regulation of viral gene expression is discussed.     

Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing alpha-N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length alpha-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human alpha-galactosidase A (alpha-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the alpha-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an approximately 35-kb insert that contained the entire alpha-GalNAc gene. A single approximately 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp alpha-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT/AG consensus rule. Analysis of 1.4 kb of 5' flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the alpha-GalNAc and the alpha-Gal A genes revealed that all six alpha-Gal A introns were identically positioned in the homologous alpha-GalNAc exonic sequence. Two additional introns, 1 and 8, were identfied in the alpha-GalNAc gene. The predicted amino acid sequences of alpha-GalNAc exons 2 through 7 and those of corresponding alpha-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of alpha-Gal A exon 7 and alpha-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the alpha-GalNAc and alpha-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.     

The evolutionary divergence of neurotransmitter receptors and second-messenger pathways

Members of the superfamily of G-protein-coupled neurotransmitter receptors have a conserved secondary structure, a moderate and reasonably steady rate of sequence change, and usually lack introns within the coding sequence. These properties are advantageous for evolutionary studies. The duplication and divergence of the genes in this gene family led to the formation of distinct neurotransmitter pathways and may have facilitated the evolution of complex nervous systems. I have analyzed this evolutionary divergence by quantitative multiple sequence alignment, bootstrap resampling, and statistical analysis of 49 adrenergic, muscarinic cholinergic, dopamine, and octopamine receptor sequences from 12 animal species. The results indicate that the first event to occur within this gene family was the divergence of the catecholamine receptors from the muscarinic acetylcholine receptors, which occurred prior to the divergence of the arthropod and vertebrate lineages. Subsequently, the ability to activate specific second-messenger pathways diverged independently in both the muscarinic and the catecholamine receptors. This appears to have occurred after the divergence of the arthropod and vertebrate lineages but before the divergence of the avian and mammalian lineages. However, the second-messenger pathways activated by adrenergic and dopamine receptors did not diverge independently. Rather, the ability of the catecholamine receptors to bind to specific ligands, such as epinephrine, norepinephrine, dopamine, or octopamine, was repeatedly modified in evolutionary history, and in some cases was modified after the divergence of the second-messenger pathways.     

Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.     

Structure of the human hemopexin gene and evidence for intron-mediated evolution

Summary The human hemopexin gene was isolated and its structure determined. The gene spans approximately 12 kb and is interrupted by nine introns. When the intron/exon pattern was examined with respect to the polypeptide segments they encode, a direct correspondence between exons and the 10 repeating units in the protein was observed. The introns are not randomly placed; they fall in the middle of the region of amino acid sequence homology in strikingly similar locations in 6 of the 10 units and in a symmetrical position in the two halves of the coding sequence. These features strongly support the hypothesis that the gene evolved through intron-mediated duplications of a primordial sequence to a five-exon cluster. A more recent gene duplication led to the present-day gene organization.