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PubMed1994

Identification of actin isoforms after in situ hydroxylamine cleavage on sequencer membranes: serum actin is a cytoplasmic isoform.

Actin was isolated from fetal bovine serum by means of affinity chromatography on DNase I-Sepharose, reversed phase chromatography and SDS-gel electrophoresis. It was identified as a cytoplasmic isoform by direct micro sequence analysis of hydroxylamine cleavage products which had been generated in situ after covalent linkage of the protein to sequencer membrane disks through arylamine or isothiocyanato functionalities.

Actins↗

PubMed1996

Pectinmethylesterase isoforms from Vigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform.

Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE gamma) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE gamma was determined. The nature protein is composed of 318 amino acids with a calculated Mtau of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in the Vmax and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.

Amino Acid Sequence↗

PubMed1994

A novel isoform of the neurofibromatosis type-1 mRNA and a switch of isoforms during murine cell differentiation and proliferation.

Four types of cDNAs encoding the GTPase-activating protein-related domain (GRD) of the mouse neurofibromatosis type-1 gene (NF1) have been cloned. One of these isoforms was a newly identified form termed type IV. Analysis of the genomic structure of the mouse NF1-GRD revealed two exons (23A and 23B) between exons 23 and 24, leading to the production of four types of NF1-GRD cDNAs by an alternative splicing mechanism. Amino-acid sequences encoded by NF1-GRD are highly conserved between human and mouse. Analysis of the expression of these transcripts in various tissues of adult mouse revealed that the type-I transcript is predominantly expressed in neural tissues such as brain and spinal cord. Other forms, termed types II, III and IV, are also expressed in various tissues. The type-I and type-II transcripts are expressed equivalently in undifferentiated P19 mouse teratocarcinoma cells, whereas type-I expression becomes predominant during neuronal differentiation by retinoic acid treatment. Expression of type I is also shown to be correlated with cessation of cell proliferation in P19 cells, but not in NIH3T3 cells. These, together with other results, suggest that the four types of NF1-GRD transcripts generated by alternative splicing have some important biological roles in cell differentiation and proliferation.

Alternative Splicing↗

PubMed2005

The metabolism of nonane, a JP-8 jet fuel component, by human liver microsomes, P450 isoforms and alcohol dehydrogenase and inhibition of human P450 isoforms by JP-8.

Nonane, a component of jet-propulsion fuel 8 (JP-8), is metabolized to 2-nonanol and 2-nonanone by pooled human liver microsomes (pHLM). Cytochrome P450 (CYP) isoforms 1A2, 2B6 and 2E1 metabolize nonane to 2-nonanol, whereas alcohol dehydrogenase, CYPs 2B6 and 2E1 metabolize 2-nonanol to 2-nonanone. Nonane and 2-nonanol showed no significant effect on the metabolism of testosterone, estradiol or N,N-diethyl-m-toluamide (DEET), but did inhibit carbaryl metabolism. JP-8 showed modest inhibition of testosterone, estradiol and carbaryl metabolism, but had a more significant effect on the metabolism of DEET. JP-8 was shown to inhibit CYPs 1A2 and 2B6 mediated metabolism of DEET, suggesting that at least some of the components of JP-8 might be metabolized by CYPs 1A2and/or 2B6.

Alcohol Dehydrogenase↗

PubMed2001

Progesterone receptor isoform A inhibits isoform B-mediated transactivation in human amnion.

Human amnion cells were transfected with progesterone receptor A and/or B, and the progesterone-dependent reporter construct, mouse mammary tumor virus promoter (MMTV), linked to a luciferase gene. In progesterone receptor B-expressing amnion that had been cultured before the onset of labour, treatment with progesterone resulted in an eightfold increase of the reporter activity, whereas in laboured cells, no such increase was seen. In contrast, progesterone receptor A was a weak activator of transcription in laboured and non-laboured amniocytes. When the isoforms A and B of the progesterone receptor were co-transfected, progesterone receptor A exhibited a marked inhibitory effect on progesterone receptor B-mediated transcription. These results show that progesterone receptors A and B function differentially, and progesterone receptor A is a transdominant repressor of progesterone receptor B-mediated transcription in human term amnion.

Amnion↗

PubMed1998

Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody.

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.

Amino Acid Sequence↗

PubMed2003

Cloning and expression of the liver and muscle isoforms of ovine carnitine palmitoyltransferase 1: residues within the N-terminus of the muscle isoform influence the kinetic properties of the enzyme.

The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases; the sequences of ovine CPT1A and CPT1B cDNAs have the accession numbers Y18387 and AJ272435 respectively and the partial adipose tissue and liver CPT1A clones have the accession numbers Y18830 and Y18829 respectively. Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid beta-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes ( CPT1A and CPT1B ). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5'-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the K (m) for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

Amino Acid Sequence↗

PubMed2000

Hypotonic cell swelling induces translocation of the alpha isoform of cytosolic phospholipase A2 but not the gamma isoform in Ehrlich ascites tumor cells.

We demonstrate that two isoforms of the cytosolic phospholipase A2, cPLA2alpha and cPLA2gamma, are present in Ehrlich ascites tumor cells. Both enzymes are almost uniformly distributed throughout the cells under control conditions, as visualized by laser-scanning confocal microscopy. Stimulation by either hypotonic cell swelling or addition of the Ca2+ ionophore A23187 results in translocation of cPLA2alpha, but not cPLA2gamma, to the nucleus, where it forms hot-spot-like clusters. Our group previously showed that release of radioactively labeled arachidonic acid, incorporated into the phospholipids of Ehrlich cells, was immediately and transiently increased on hypotonic cell swelling [Thoroed, S.M., Lauritzen, L., Lambert, I.H., Hansen, H.S. & Hoffmann, E.K. (1997) J. Membr. Biol. 160, 47-58]. We now demonstrate that arachidonic acid is released from the nuclear fraction following hypotonic exposure. Stimulation of Ehrlich cells with A23187 also leads to an increase in arachidonic acid release from the nucleus. However, as hypotonic cell swelling is not accompanied by any detectable increase in intracellular concentration of free cytosolic Ca2+ ([Ca2+]i), stimulus-induced translocation of cPLA2alpha can also occur without elevation of [Ca2+]i. The stimulus-induced translocation of cPLA2alpha appears not to be prevented by inhibition of mitogen-activated protein (MAP) kinase activation, p38 MAP kinase, tyrosine kinases and protein kinase C, hence, phosphorylation is not crucial for the stimulus-induced translocation of cPLA2alpha. Disruption of F-actin did not affect the translocation process, thus, an intact F-actin cytoskeleton does not seem to be required for translocation of cPLA2alpha.

Animals↗

PubMed1995

Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.

The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.

Animals↗

PubMed1996

Expression of a cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT I). M-CPT I is the predominant CPT I isoform expressed in both white (epididymal) and brown adipocytes.

We set out to determine if the cDNA encoding a carnitine palmitoyltransferase (CPT)-like protein recently isolated from rat brown adipose tissue (BAT) by Yamazaki et al. (Yamazaki, N., Shinohara, Y., Shima, A., and Terada, H. (1995) FEBS Lett. 363, 41-45) actually encodes the muscle isoform of mitochondrial CPT I (M-CPT I). To this end, a cDNA essentially identical to the original BAT clone was isolated from a rat heart library. When expressed in COS cells, the novel cDNA and our previously described cDNA for rat liver CPT I (L-CPT I) gave rise to products with the same kinetic characteristics (sensitivity to malonyl-CoA and Km for carnitine) as CPT I in skeletal muscle and liver mitochondria, respectively. When labeled with [3H]etomoxir, recombinant L-CPT I and putative M-CPT I, although having approximately the same predicated masses (88.2 kDa), migrated differently on SDS gels, as did CPT I from liver and muscle mitochondria. The same was true for the products of in vitro transcription and translation of the L-CPT I and putative M-CPT I cDNAs. We conclude that the BAT cDNA does in fact encode M-CPT I. Northern blots using L- and M-CPT I cDNA probes revealed the presence of L-CPT I mRNA in liver and heart and its absence from skeletal muscle and BAT. M-CPT I mRNA, which was absent from liver, was readily detected in skeletal muscle and was particularly strong in heart and BAT. Whereas the signal for L-CPT I was more abundant than that for M-CPT I in RNA isolated from whole epididymal fat pad, this was reversed in purified adipocytes from this source. These findings, coupled with the kinetic properties and migration profiles on SDS gels of CPT I in brown and white adipocytes, indicate that the muscle form of the enzyme is the dominant, if not exclusive, species in both cell types.

Adipocytes↗

PubMed1998

Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation.

Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.

Cyclic AMP-Dependent Protein Kinases↗

PubMed1993

TCR isoform containing the Fc receptor gamma chain exhibits structural and functional differences from isoform containing CD3 zeta.

The structure and function of the TCR-CD3 complex containing a homodimer of the gamma chain of the high affinity receptor for IgE (FcR gamma) (FcR gamma+ TCR) was investigated by transfecting the FcR gamma gene into a CD3 zeta-, CD3 eta-, FcR gamma- T cell line. Introduction of FcR gamma, as well as CD3 zeta, induced a high expression of the TCR-CD3 complex on the cell surface. Transfected FcR gamma formed a homodimer and associated firmly with the TCR alpha beta dimer but only weakly with the CD3 gamma delta epsilon. Stimulation of both FcR gamma and CD3 zeta transfectants by antibodies against TCR or CD3 induced accumulation of inositol phosphates, the Ca2+ response, IL-2 production, and growth inhibition. On the other hand, antigen stimulation of transfectants expressing FcR gamma as well as CD3 zeta induced IL-2 production, but only the latter exhibited the antigen-induced growth inhibition. In vitro kinase assay suggested that the CD3 zeta dimer but not the FcR gamma dimer associates with the Fyn kinase. These results indicate that the FcR gamma homodimer is able to form a functional TCR complex but that the mode of assembly and the signaling function of FcR gamma+ TCR, including its association with tyrosine kinase(s), may differ from the TCR-CD3 complex containing CD3 zeta homodimers (zeta+ TCR). This provides an example which illustrates that different TCR isoforms mediate distinct signals and functions.

Animals↗

PubMed2004

The Na+/H+ exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation.

The Na(+)/H(+) exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (-/-) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (-/-) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pH(i)), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pH(i). Crypts isolated from NHE2 (-/-) mice displayed increased acid-activated NHE1- and NHE3-attributable Na(+)/H(+) exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (-/-) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pH(i) and volume homeostasis.

Acids↗

PubMed2002

Selective regulation of glutamic decarboxylase isoform 65, but not isoform 67, in the bed nucleus of the stria terminalis and the preoptic area of the ewe brain across the estrous cycle.

gamma-Aminobutyric acid neurons in the preoptic area (POA) of the brain may regulate GnRH neurons. The level of expression of two isoforms (65 and 67) of glutamic acid decarboxylase (GAD) in the ewe brain was determined across the estrous cycle by in situ hybridization. GAD mRNA expression (cell number and silver grains/cell) was examined in the subdivisions of the bed nucleus of stria terminalis (BnST), in the diagonal band of Broca, and the POA. The number of cells expressing GAD(65) and GAD(67) mRNA did not change across the cycle. Within the rostro-dorsal BnST, the number of silver grains/cell for GAD(65) mRNA was lower in the follicular phase than the luteal phase or at estrus. In the rostro-lateral division, expression was lower in the follicular phase. In the POA, the number of silver grains/cell for GAD(65) mRNA was lower at estrus than during the luteal phase. The number of silver grains/cell for GAD(67) mRNA did not change across the estrous cycle. GAD(65) is thought to be the active enzyme during periods of high demand of GABA and our results are consistent with the GABA neurons of BnST being most active during the luteal phase of the estrous cycle.

Animals↗

PubMed1987

Apolipoprotein E isoform phenotyping methodology and population frequency with identification of apoE1 and apoE5 isoforms.

Minigel methodology has been utilized for apolipoprotein (apo) E isoform phenotyping and criteria for distinguishing the apoE4/4 phenotype (mean apoE4/apoE3 ratio: 5.81) from the apoE4/3 phenotype (mean ratio: 1.01) and the apoE3/3 phenotype (mean apoE3/apoE2 ratio: 2.67) from the apoE3/2 phenotype (mean ratio: 0.76) based on gel scanning were developed. ApoE allele frequencies in 1209 subjects were: apoE3, 0.786; apoE4, 0.135; apoE2, 0.075; apoE5, 0.002; and apoE1, 0.002. Subjects with the apoE2 allele tended to have higher plasma very low density lipoprotein (VLDL) cholesterol and lower low density lipoprotein (LDL) cholesterol concentrations than subjects with the apoE3 allele, while the converse was true for subjects with the apoE4 allele. Subjects with the rare apoE1 allele had values similar to those with the apoE2 allele, while subjects with the rare apoE5 allele had values similar to those with the apoE4 allele.

Adult↗

PubMed1996

Isoform-specific sensitization of adenylyl cyclase activity by prior activation of inhibitory receptors: role of beta gamma subunits in transducing enhanced activity of the type VI isoform.

Many different types of cells develop increased adenylyl cyclase activity (sensitization) on prior treatment with drugs such as opiates that acutely inhibit the enzyme. We found that human embryonic kidney (HEK) 293 m2 cells, which express the inhibitory m2 muscarinic cholinergic receptor, exhibit a large increase in forskolin-stimulated cAMP synthesis when the cells are preincubated with the muscarinic agonist carbachol for > or = 5 min and forskolin stimulation is performed in the presence of the muscarinic antagonist atropine. To determine whether a specific isoform of adenylyl cyclase is susceptible to the adaptation induced by prior activation of inhibitory receptors, cells were transfected with expression vectors encoding adenylyl cyclase types I, II, and VI, representing three major groups of the adenylyl cyclase family. Preincubation of the cells with carbachol for 30 min resulted in a significant increase in prostaglandin E1-stimulated cAMP accumulation in cells expressing type VI, but not type I or type II, adenylyl cyclase. A similar selective increase in activity from type VI adenylyl cyclase was observed for prior treatment with the D2 dopamine agonist quinpirole and stimulation of cAMP synthesis with human chorionic gonadotropin in cells transfected with expression vectors coding for the cognate receptors. We next investigated whether beta gamma subunits play a role in the sensitization of type VI adenylyl cyclase activity; using expression of alpha tau to inhibit beta gamma-mediated effects, we found that the quinpirole-induced sensitization of type VI adenylyl cyclase was abolished. However, beta gamma subunits do not seem to directly activate type VI adenylyl cyclase, in contrast with their ability to directly activate the type II enzyme. Therefore, beta gamma subunits liberated after activation of inhibitory receptors seem to indirectly cause an increase in activity of type VI adenylyl cyclase. Indirect activation of the type VI enzyme by beta gamma subunits is a novel mechanism contributing to the sensitization of adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1997

Mouse IL-1 receptor antagonist isoforms: complementary DNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1 receptor antagonist in vivo.

IL-1R antagonist (IL-1Ra) is a competitive inhibitor of the binding of IL-1 to IL-1R. IL-1Ra refers to two different proteins derived from the same gene by alternate splicing of two different first exons. One protein contains a leader sequence and is secreted (sIL-1Ra), whereas the other remains intracellular (icIL-1Ra). We describe the cloning of mouse icIL-1Ra cDNA, the expression of the recombinant mouse icIL-1Ra protein, and the tissue distribution of sIL-1Ra and icIL-1Ra mRNA and of icIL-1Ra protein in control and LPS-injected mice. As described in the human and the rabbit, mouse icIL-1Ra protein differs from mature mouse sIL-1Ra protein by seven amino acids at the amino terminus. In addition, human and mouse icIL-1Ra are 77% identical. Regulation of IL-1Ra isoforms was examined in normal mice and after LPS injection. Circulating levels were undetectable in control mice, but were strongly increased 4 h after LPS injection. Using a ribonuclease protection assay (RPA), we found that icIL-1Ra mRNA was expressed constitutively in skin and in LPS-stimulated RAW 264.7 murine macrophages. Consistent with the RNA studies, Western blot analysis showed that murine icIL-1Ra protein was constitutively expressed in skin and in LPS-stimulated RAW 264.7 cells. In contrast, sIL-1Ra mRNA was not detected by RPA in tissues of control mice, but was strongly up-regulated in the lung, spleen, and liver after LPS injection. Using RPA, primer extension assay and 5' rapid amplification of cDNA ends, we were able to demonstrate the presence of different transcription start sites for murine sIL-1Ra mRNA.

Alternative Splicing↗

PubMed2006

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms.

The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the P. falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine-sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification by a Dd2 allele expressing the T76K "back mutation" was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca(2+) stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

Animals↗