Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.     

ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily

Using differential display of rat fetal and postnatal cardiomyocytes, we have identified a novel seven-transmembrane receptor, ETL. The cDNA-predicted amino acid sequence of ETL indicated that it encodes a 738-aa protein composed of a large extracellular domain with epidermal growth factor (EGF)-like repeats, a seven-transmembrane domain, and a short cytoplasmic tail. ETL belongs to the secretin family of G-protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter are characterized by a variable number of extracellular EGF and cell surface domains and conserved seven transmembrane-spanning regions. ETL mRNA expression is up-regulated in the adult rat and human heart. In situ hybridization analyses revealed expression in rat cardiomyocytes and abundant expression in vascular and bronchiolar smooth muscle cells. In COS-7 cells transfected with Myc-tagged rat ETL, rat ETL exists as a stable dimer and undergoes endoproteolytic cleavage of the extracellular domain. The proteolytic activity can be abolished by a specific mutation, T455A, in this domain. In transfected mammalian cells, ETL is associated with cell membranes and is also observed in cytoplasmic vesicles. ETL is the first seven-transmembrane receptor containing EGF-like repeats that is developmentally regulated in the heart.     

Identification and characterization of human taste receptor genes belonging to the TAS2R family

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.     

Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars [1, 2]. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor [3, 4], and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor [5-7]. We carried out molecular analyses of mutations in Tre to elucidate their involvement in the gustatory phenotype. Here, we show that Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression was not always associated with mutations in Tre. In flies with the spontaneous mutation Tre(01), both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre(01). Taken together, the mutation analyses support that Gr5a is allelic to Tre.     

Role of rhodopsin N-terminus in structure and function of rhodopsin-bitter taste receptor chimeras

The bitter taste receptors (T2Rs) belong to the G protein-coupled receptor (GPCR) superfamily. In humans, bitter taste sensation is mediated by 25 T2Rs. Structure–function studies on T2Rs are impeded by the low-level expression of these receptors. Different lengths of rhodopsin N-terminal sequence inserted at the N-terminal region of T2Rs are commonly used to express these receptors in heterologous systems. While the additional sequences were reported, to enhance the expression of the T2Rs, the local structural perturbations caused by these sequences and its effect on receptor function or allosteric ligand binding were not characterized. In this study, we elucidated how different lengths of rhodopsin N-terminal sequence effect the structure and function of the bitter taste receptor, T2R4. Guided by molecular models of T2R4 built using a rhodopsin crystal structure as template, we constructed chimeric T2R4 receptors containing the rhodopsin N-terminal 33 and 38 amino acids. The chimeras were functionally characterized using calcium imaging, and receptor expression was determined by flow cytometry. Our results show that rhodopsin N-terminal 33 amino acids enhance expression of T2R4 by 2.5-fold and do not cause perturbations in the receptor structure.     

Alternative transcripts of the rat and human dopamine D3 receptor

A cDNA for the rat dopamine D3 receptor containing a 113 bp deletion has been isolated. The segment deleted, encompassing the first extracellular loop and third transmembrane domain, alters the reading frame, introducing 19 amino acids not found in the full length receptor followed by a premature stop codon. This novel mRNA encodes a 109 amino acid protein containing two putative transmembrane domains. A similar variant cDNA for the human D3 receptor has also been identified.     

Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii

The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.     

Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus.

Glvr1 encodes the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related gene Glvr2 encodes the human receptor for amphotropic murine leukemia viruses (A-MLVs). The two proteins are 62% identical in their amino acid sequences and are predicted to have 10 transmembrane domains and five extracellular loops. A stretch of nine amino acids (region A) in the predicted fourth extracellular loop was previously shown to be critical for the function of Glvr1 as receptor for GALV and FeLV-B. Glvr1 and -2 show clusters of amino acid differences in several of their predicted extracellular loops, with the highest degree of divergence in region A. Chimeras were made between the two genes to further investigate the role of Glvr1 region A in defining receptor specificity for GALV and FeLV-B and to map which regions of Glvr2 control receptor specificity for A-MLVs. Region A from Glvr1 was sufficient to confer receptor specificity for GALV upon Glvr2, with the same chimera failing to act as a receptor for FeLV-B. However, introduction of additional N- or C-terminal Glvr1-encoding sequences in addition to Glvr1 region A-encoding sequences resulted in functional FeLV-B receptors. Therefore, FeLV-B is dependent on Glvr1 sequences outside region A for infectivity. The receptor specificity of Glvr2 for A-MLV could not be mapped to a single critical region; rather, N-terminal as well as C-terminal Glvr2-encoding sequences could confer specificity for A-MLV infection upon Glvr1. Surprisingly, though GALV/FeLV-B and A-MLV belong to different interference groups, some chimeras functioned as receptors for all three viruses.     

Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells

Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca(2+). CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

Molecular cloning, sequence and expression of follicle-stimulating hormone receptor in the lizard Podarcis sicula

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.     

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.     

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.