Construction of chimeras between human VIP1 and secretin receptors: identification of receptor domains involved in selectivity towards VIP, secretin, and PACAP.
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Animals↗
Biomedical subjects
K Du
Publications and source records attributed to K Du.
CREB is a regulatory target for the protein kinase Akt/PKB.
The nuclear factor CREB stimulates the expression of cellular genes following its protein kinase A-mediated phosphorylation at Ser-133. Ser-133 phosphorylation, in turn, activates target gene expression by promoting recruitment of the co-activator CBP. Recent studies showing that CREB and its paralog CREM are required for survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the growth factor-dependent Ser/Thr kinase Akt/PKB. When overexpressed in serum-stimulated cells, Akt/PKB potently induced Ser-133 phosphorylation of CREB and promoted recruitment of CBP. Correspondingly, Akt/PKB stimulated target gene expression via CREB in a phospho(Ser-133)-dependent manner. Akt/PKB induced CREB activity only in response to serum stimulation, and this effect was suppressed by the phosphatidylinositol 3-kinase inhibitor LY 294002. Our results support the notion that Akt/PKB promotes cell survival, at least in part, by stimulating the expression of cellular genes via the CREB/CBP nuclear transduction pathway.
Cell Line↗
The Gfi-1B proto-oncoprotein represses p21WAF1 and inhibits myeloid cell differentiation.
Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1. Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at -1518 to -1530 in the p21WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21WAF1 and inhibited G1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of the p21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21WAF1 induction by IL-6.
3T3 Cells↗
Site-directed mutagenesis of human vasoactive intestinal peptide receptor subtypes VIP1 and VIP2: evidence for difference in the structure-function relationship.
Vasoactive intestinal peptide (VIP1 and VIP2) receptors belong to the new class II subfamily of G protein-coupled receptors. We investigated here human VIP1 and VIP2 receptors by mutating in their extracellular domains all amino acid residues that are conserved in VIP receptors but are different in other members of their subfamily. They are present in 1) the N-terminal domain, i.e., E36, I43, S64, D132 and F138 in the VIP1 receptor and E24, I31, S53, D116 and F122 in the VIP2 receptor; 2) the second extracellular loop, i.e., T288 and S292 in the VIP1 receptor and T274 and S278 in the VIP2 receptor. These residues were changed to alanine (A), and cDNAs were transfected into Cos cells. For the VIP1 receptor, no specific 125I-VIP binding could be detected in cells transfected with the E36A mutant, whereas other mutants exhibited Kd values similar to that of the wild-type receptor, with the exception of S64A, for which a 3-fold increase of Kd was observed. For the VIP2 receptor, no specific 125I-VIP binding could be observed with the E24A mutant, whereas other mutants exhibited dissociation constants similar to that of the wild-type receptor, with the exception of I31A and T274A mutants, for which a 11- and 5-fold increase of Kd was observed, respectively. cAMP production experiments provided evidence that the E36A VIP1 receptor and the E24A VIP2 receptor mutants mediated almost no response upon VIP exposure. For the I31A and T274A mutants of the VIP2 receptor and the S64A mutant of the VIP1 receptor, the EC50 values of VIP for stimulating cAMP production were increased 35, 8 and 3 times as compared with that observed for the wild-type receptor, respectively. Immunofluorescence studies indicated that all mutants were normally expressed by Cos cells. These data provide the first evidence for differences in the structure-function relationship of VIP1 and VIP2 receptors.
Amino Acid Sequence↗
Alternative splicing and structure of the human and mouse SFRS5/HRS/SRp40 genes.
HRS/SRp40/SFRS5 (HRS) is an SR (serine-arginine-rich) protein which regulates both alternative splicing and basal splicing. HRS mRNA contains several transcripts, including HRS-SF and HRS-LF which have different temporal patterns of expression in proliferating liver. As previously reported, HRS-SF mRNA encodes the SR splicing factor. However, the identity of HRS-LF remained unknown. Here, we cloned and characterized the mouse HRS gene, partial human HRS gene, and several cDNAs derived from HRS-LF mRNA. The mouse HRS gene spans 5050 bp and contains eight exons and seven introns. HRS-LF mRNA contains a 1.2 kb insert within the SF mRNA with stop codons in all three reading frames. A comparison of HRS-LF and the HRS gene revealed that HRS-LF mRNA is an intron-retaining product which contains intron 5. At most, HRS-LF encodes a truncated HRS protein with one RNA binding domain. Interestingly, intron 5 demonstrates 90% identity between the mouse and human HRS genes, implying that intron 5 might play an important role in regulating HRS gene splicing or expression.
Alternative Splicing↗
Expression analysis and chromosomal assignment of the human SFRS5/SRp40 gene.
Alternative splicing plays a major role in the regulation of gene expression. SFRS5/SRp40 is a member of the serine/arginine (SR) protein family of regulators of alternative pre-mRNA splicing. We cloned the human SFRS5 cDNA and observed two major SFRS5 transcripts, an approximately 1.8-kb short form and an approximately 3.3-kb long form, in both human and rat tissues. Both transcripts were detected in all human tissues examined, but there were notable tissue-specific differences in their relative abundance, the short form being most abundant in retina. Affinity-purified SFRS5 antisera recognized a single 40-kDa polypeptide in human and mouse retinal lysates. The abundant retinal expression of SFRS5 was not restricted to any specific cell type, since immunofluorescent labeling of human retinal sections identified the SFRS5 protein in nuclei of all three nuclear layers of the retina. The human SFRS5 gene was localized to human chromosome 14q24 by fluorescence in situ hybridization and PCR analysis of a human/hamster somatic cell hybrid panel.
Alternative Splicing↗
Aspartate 196 in the first extracellular loop of the human VIP1 receptor is essential for VIP binding and VIP-stimulated cAMP production.
VIP receptors belong to a subfamily of G protein-coupled receptors that includes secretin, glucagon, PTH and several other receptors. We have used site-directed mutagenesis to investigate the requirement of some highly conserved residues in the extracellular loops including aspartate 196 (mutant D196A), leucine 199 (mutant L199A), tryptophane 286 (mutant W286A) and tryptophane 294 (mutant W294A) for the ability of the human VIP1 receptor to bind VIP and to mediate VIP-stimulated cAMP production. After transfection of mutated cDNAs in Cos-7 cells, it appeared that 1) mutants L199A, W286A and W294A bound VIP with the same dissociation constant as the wild-type receptor whereas mutant D196A did not bind 125I-VIP; 2) mutants L199A, W286A and W294A mediate VIP-stimulated cAMP production with the same EC50 as the wild-type receptor whereas VIP displayed a 500-fold decrease of potency in promoting cAMP production through mutant D196A. Since all mutated receptor proteins were expressed and delivered at the plasma membrane (immunofluorescence studies), it is concluded that the first extracellular loop of the human VIP1 receptor contains a highly conserved aspartate residue which is essential for VIP binding and VIP-stimulated cAMP production.
Amino Acid Sequence↗
HRS/SRp40-mediated inclusion of the fibronectin EIIIB exon, a possible cause of increased EIIIB expression in proliferating liver.
Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.
Alternative Splicing↗
Improved methods for making and using glass knives.
We have observed over time that the right side of a glass knife is the optimal cutting edge for microtomy if the counterpiece (heel opposite the edge) is controlled within 1 mm. The right cutting edge has been considered the "saw toothed" side and has not been used for ultrathin sectioning. We have observed that the right cutting edge is sharper and more durable than the left. Light and scanning electron microscopy were used to observe the cutting edge, and transmission electron microscopy was used to examine semithin and ultrathin sections of animal and plant tissues cut by the right and left sides of the cutting edge. The results indicate that the cutting edge becomes sharper and more durable from left to right. Both the quality and efficiency of ultrathin sectioning is improved by using the right cutting edge.
Animals↗
[Directed mutagenesis of human recombinant receptors of vasoactive intestinal peptide of VIP1 and VIP2 type. Difference in structure-activity relationship].
OBJECTIVES: Two vasoactive intestinal peptide (VIP) receptor subtypes have been cloned. We studied the structure-function relationship of human VIP1 and VIP2 receptors by mutating residues specifically conserved in extracellular domains of these receptors: N-terminal domain (E36, I43, S64, D132 and F138 in VIP1 receptor corresponding to E24, I31, S53, D116 and F122 in VIP2 receptor) and second loop (T288 and S292 in VIP1 receptor corresponding to T274 and S278 in VIP2 receptor). METHODS: Residues were mutated into alanine (A) and the corresponding cDNAs were transfected into Cos cells. Wild-type and mutated receptors were characterized in transfected cells by ligand binding assay using 125I-VIP and cAMP measurements upon VIP challenge. RESULTS: Regarding the VIP1 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E36A mutant whereas other mutants, with the exception of S64A, exhibited dissociation constants similar to that of the wild-type receptor. The S64A mutant showed a 3-fold increase of its dissociation constant as compared to the wild-type receptor. cAMP experiments showed that the E36A mutant mediated a very weak stimulation by VIP. Regarding the VIP2 receptor, no specific binding of 125I-VIP could be detected on Cos cells transfected with the E24A. I31A and T274A mutants whereas all other mutants exhibited dissociation constants similar to that of the wild-type receptor. cAMP experiments showed that the E24A mutant mediated a very weak stimulation by VIP. Regarding I31A and T274A mutants, the EC50 values were increased 10 and 50 times as compared to the wild-type receptor, respectively. CONCLUSION: a) The conserved glutamate (E) residue in the N-terminal domain of VIP1 and VIP2 receptors is crucial for VIP binding; b) The VIP2 receptor contains two conserved residues isoleucine 31 and threonine 274 which are critical for VIP binding while they can be mutated without loss of function in the VIP1 receptor. This difference in the structure-function relationship should be instrumental for the development of a selective pharmacology of VIP receptor subtypes.
Alanine↗
[The design of an OEM module-based hand-hold pulse oximeter].
This paper describes the software and hardware design of an OEM module-based hand-hold pulse oximeter. The instrument has the features of compact structure, reliable performance, having all the necessary functions, providing measurements on any patients and low power consumption. It is possible to make this instrument as a commercial product in shorter period.
Equipment Design↗
[The cloning and expression of human secreted interleukin 2 receptor alpha chain gene in eukaryotic cells].
By deleting the sequences encoding the cytoplasmic domain, transmembrane domain and the 5' untranslated region, human interleukin-2 receptor alpha cDNA fragment was subcloned into an eukaryotic expression vector pRc/CMV. The recombinant plasmid was then transfected into CHO cells by either electroporation or calcium phosphate precipitation. 11 G418-resistent clones were randomly selected for detection of mRNA expression. 5 clones out of the 11 were proved to be positively hybridyzed with IL-2R alpha cDNA probe. All of the 5 positive cell lines also expressed sIL-2R alpha in their supernatants as detected by ELISA. The establishment of rhsIL-2R alpha cell lines is of importance in the detection and purification of IL-2 based on the ability of affinity binding between IL-2 and its recombinant receptor.
Animals↗
Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing.
We have identified 41 novel and many previously known growth response genes induced in regenerating liver and insulin-treated Reuber H35 cells, a rat hepatoma cell line that grows in response to physiologic concentrations of insulin and retains some properties of regenerating liver. Although many genes are expressed similarly in the two systems, there are important differences in the kinetics of induction of some genes. These differences allowed us to identify and characterize novel genes that are highly insulin-induced and expressed as delayed-early genes in regenerating liver. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. Sequence analysis of HRS, a highly insulin-induced delayed-early gene, demonstrated that it is a member of the family of regulators of alternative pre-mRNA splicing. Different forms of HRS mRNA are temporally regulated during the growth response, suggesting that HRS could autoregulate processing of its pre-mRNA. Given the dramatic increase in RNA production during late G1, proteins induced by mitogens like insulin that control RNA processing are likely to have important roles in cell cycle regulation.
Alternative Splicing↗