Identification of amino acids in the N-terminal domain of corticotropin-releasing factor receptor 1 that are important determinants of high-affinity ligand binding

The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76-84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg76 or Asn81 but not Gly83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100-1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding.     

Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: implications for the ligand-binding site

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg76, Asn81, Gly83, Leu88, and Ala89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln76, Gly81, Val83, His88, and Leu89) resulted in a receptor that had approximately 30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (approximately 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg76, Asn81, and Gly83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln76, Gly81, and Val83) resulted in a receptor with approximately 11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.     

Macrophage inhibition of lymphocyte and tumor cell growth is mediated by 25-hydroxycholesterol in the cell membrane

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (approximately 98%) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (approximately 97%). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the N-terminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (KD = 1.28 nM), human/rat CRF (KD = 1.09 nM), urocortin (KD = 0.37 nM) and sauvagine (KD = 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (KD = 12.4 nM). In agreement with the binding data half maximum effective EC50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1.     

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) 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 mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Complete cDNA sequence, genomic structure, and chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26

The lpA1/Gpcr26 locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3' rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5' sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor, Edg1, but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 in Mus spretus, and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for the vzg-1/lpA1 subfamily of receptors compared to those of homologous orphan receptor genes.     

Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.     

The novel UGT1 gene complex links bilirubin, xenobiotics, and therapeutic drug metabolism by encoding UDP-glucuronosyltransferase isozymes with a common carboxyl terminus

The UDP-glucuronosyltransferase system (transferase) plays an important role in the pharmacokinetics of clearance of endogenous metabolites, therapeutic drugs, and xenobiotics. The human bilirubin and phenol transferases are encoded by the same gene complex which we designate UGT1. The gene arrangement indicates there are 6 exon 1s each with a promoter and each of which can predictably undergo differential splicing to the 4 common exons (2 through 5) to generate possibly 6 different mRNAs. The entire unique amino acid terminus of each isoform is encoded by an exon 1, and the common carboxyl terminus is encoded by the 4 common exons. Evidence supports the existence of other exon 1s upstream of the currently described locus. The 13-bp deletion in exon 2 represents the most common defect, to date, in the Crigler-Najjar, Type I individuals. Different point mutations in the 4 common exons and in exon 1 of UGT1A, however, also account for defective bilirubin transferase activity. The gene arrangement, in conjunction with the toxicity data from the Gunn rat, leads to the prediction that detoxification of bilirubin, xenobiotics, and therapeutic drugs is linked to the UGT1 locus. The Crigler-Najjar syndromes are uncommon, but the Gilbert individuals are commonly represented in 6% of the population. It is expected that, similar to the deleterious mutations in the common region of the UGT1 locus in Crigler-Najjar, Type I individuals, there is a range of moderate to intermediate deleterious mutations in this region of the gene of at least some Gilbert's individuals. Linkages, therefore, at this locus could signal that these individuals are at risk for certain drug toxicities and/or idiosyncratic drug reactions.     

Lack of association between ferritin level and measures of LDL oxidation: the ARIC study. Atherosclerosis Risk in Communities

The neprilysin gene is composed of three distinct 5' noncoding exons which can be joined to the first coding exon to generate multiple mRNA species, all encoding the same protein. Genomic fragments containing upstream sequences of each of these three noncoding exons from the rat neprilysin gene were subcloned in the promoterless vector pXp1, which contains the luciferase reporter gene. Expression was compared between a neprilysin positive human spinal cord cell line, HSC-2, and neprilysin negative lines MCF-7, a human breast adenocarcinoma cell line and Hep G2, a human liver carcinoma cell line. The first and second promoter regions showed high activity in the positive cell line, but low activity in the negative cell lines. An analysis of the exon 1 promoter region showed that the proximal 85 nucleotides exhibited basal promoter activity. An enhancer-like sequence was found to be located within a 22-bp fragment located at -136 to -115. Scanning mutagenesis of a 29-bp fragment containing the enhancer-like sequence showed that changes in each 5- or 6-bp segment throughout this fragment decreased activity; however, mutations of the segment encompassing positions 19 to 24 eliminated >98% of the promoter activity. Binding of nuclear proteins from HSC-2 cells to this 29-bp fragment was observed by gel shift analysis. The ability of mutations within the 29-bp fragment to affect enhancer activity correlated with the ability of these mutant oligonucleotides to compete for the wild-type sequence in gel shift assays.     

Genomic organization of a human killer cell inhibitory receptor gene

We have cloned a region of human chromosome 19q13.4 which contains multiple killer cell inhibitory receptor (KIR) loci. By random and directed sequence analysis of these KIR-specific clones, we deduced the genomic structure of KIR genes. A locus encoding a member of the NKAT-2 family of KIRs is presented here. The structure of the gene is reminiscent of loci of the Fc receptor gene family, and the two sets of genes may derive from a common ancestor. The KIR gene contains potentially nine exons. The first two exons encode the leader sequence, as in Fc receptor genes. The third exon encodes an untranslated pseudo exon specifying an immunoglobulin domain with an in-frame stop codon. Expressed cDNAs do not contain this exon. This finding is consistent with the hypothesis that certain KIR genes may have been derived from the duplication of a primordial three immunoglobulin domain structure with subsequent skipping of one exon to derive genes with two expressed immunoglobulin domains. Variation in numbers of immunoglobulin domains in different KIR genes is facilitated by conservation of splicing frame in respect to the codon triplet for each immunoglobulin domain.