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PubMed2005

Developmental stage-selective effect of somatically mutated leukemogenic transcription factor GATA1.

Acquired mutations in the hematopoietic transcription factor GATA binding protein-1 (GATA1) are found in megakaryoblasts from nearly all individuals with Down syndrome with transient myeloproliferative disorder (TMD, also called transient leukemia) and the related acute megakaryoblastic leukemia (DS-AMKL, also called DS-AML M7). These mutations lead to production of a variant GATA1 protein (GATA1s) that is truncated at its N terminus. To understand the biological properties of GATA1s and its relation to DS-AMKL and TMD, we used gene targeting to generate Gata1 alleles that express GATA1s in mice. We show that the dominant action of GATA1s leads to hyperproliferation of a unique, previously unrecognized yolk sac and fetal liver progenitor, which we propose accounts for the transient nature of TMD and the restriction of DS-AMKL to infants. Our observations raise the possibility that the target cells in other leukemias of infancy and early childhood are distinct from those in adult leukemias and underscore the interplay between specific oncoproteins and potential target cells.

Adult↗
PubMed2000

Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia.

Initiation of skin tumors in mice is associated with the formation of oncogenic mutations in the H-ras gene. Mice treated on the dorsal skin with the potent polycyclic aromatic hydrocarbon (PAH) carcinogen dibenzo[a,l]pyrene (DB[a,l]P) form papillomas carrying the H-ras codon 61 (CAA to CTA) mutations. These mutations are induced in early preneoplastic skin within 1 day after DB[a,l]P treatment (Oncogene 16 (1998) 3203-3210) and appear to be related to DB[a,l]P-Ade-depurinating adducts (Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10422-10426). The rapid kinetics of mutation induction suggests that abasic sites generated from base depurination may undergo error-prone excision repair in pre-S-phase cells to induce these mutations. Analysis of mutations in the H-ras exon 1 and 2 region in DB[a,l]P-treated early preneoplastic skin indicated great changes in mutation spectra in the preneoplastic period. The initial spectra contained abundant A-->G mutations, which frequently occurred 3' to a putative conserved sequence (TGN-doublet). These mutations appeared to be induced initially as mismatched (G.T) heteroduplexes and then converted into double-stranded mutations by one round of replication. Unlike the A-->G mutations found in DB[a, l]P-treated skin (which forms 99% depurinating adducts), A-->G mutations found in anti-DB[a,l]P-diol epoxide-treated skin (forms 97% stable adducts) did not appear to be G.T heteroduplexes. These results, therefore, suggest that under these conditions, the repair errors occurred only from abasic sites but not from stable adducts. Initiated cells carrying specific oncogenic mutations, formed presumably by misrepair, underwent rapid clonal expansion and regression (transient clonoplasia). The multiplication of initiated stem cells during transient clonoplasia may be a factor determining the tumor-initiating potential of some PAH carcinogens.

Animals↗
PubMed1987

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

Adult↗
PubMed2004

Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.

Aminopeptidases↗
PubMed2001

The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

Animals↗
PubMed1996

Genetic complementation between replication-defective mutants of HIV-1 and SIVagm.

To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses.

Animals↗
PubMed2001

Prevalence, phenotypic spectrum, and modes of inheritance of gonadotropin-releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism.

Mutations in the GnRH receptor (GNRHR) have been described as a cause of reproductive failure in a subset of patients with idiopathic hypogonadotropic hypogonadism (IHH). Given the apparent rarity of these mutations, we set out to determine the frequency and distribution of GNRHR mutations in a heterogeneous population of patients with IHH who were well characterized with respect to diagnosis, phenotype, and mode of inheritance and to define their distribution within the receptor protein. One hundred and eight probands with IHH were screened for mutations in the coding sequence of GNRHR. Forty-eight of the 108 patients had a normal sense of smell, whereas the remaining 60 had anosmia or hyposmia (Kallmann syndrome). Exon segments in the GNRHR were screened for mutations using temperature gradient gel electrophoresis, and all mutations were confirmed by direct sequencing. Five unrelated probands (3 men and 2 women), all normosmic, were documented to have changes in the coding sequence of the GNRHR. Two of these probands were from a subgroup of 5 kindreds consistent with a recessive mode of inheritance, establishing a GNRHR mutation frequency of 2 of 5 (40%) in patients with normosmic, autosomal recessive IHH. The remaining 3 probands with GNRHR mutations were from a subgroup of 18 patients without evidence of familial involvement, indicating a prevalence of 3 of 18 (16.7%) in patients with sporadic IHH and a normal sense of smell. Among the five individuals bearing GNRHR mutations, a broad spectrum of phenotypes was noted, including testicular sizes in the male that varied from prepubertal to the normal adult male range. Three probands had compound heterozygous mutations, and two had homozygous mutations. Of the eight DNA sequence changes identified, four were novel: Thr(32)Ile, Cys(200)Tyr, Leu(266)Arg, and Cys(279)TYR: COS-7 cells transiently transfected with complementary DNAs encoding the human GNRHR containing each of these four novel mutations failed to respond to GnRH agonist stimulation. We conclude that 1) the spectrum of phenotypes in patients with GNRHR mutations is much broader than originally anticipated; 2) the frequency of GNRHR mutations may be more common than previously appreciated in familial cases of normosmic IHH and infrequent in sporadic cases; and 3) functional mutations of the GNRHR are distributed widely throughout the protein.

Amino Acid Sequence↗
PubMed2006

GATA1 mutations in acute leukemia in children with Down syndrome.

It has been reported that somatic mutations in the X-linked GATA1 gene are present in hematological clonal disorders in children with Down syndrome (DS). We analyzed retrospective samples of DS children with acute myeloid leukemia, transient leukemia (TL), and myelodysplastic syndrome (MDS) to test whether the specificity of GATA1 mutations can be helpful in distinguishing these hematopoietic disorders. A total of 49 samples were subjected to GATA1 mutation screening by direct sequencing and denaturing polyacrylamide gel electrophoresis (PAGE). Mutations in exon 2 of GATA1 were detected in six of eight DS-AML M7 samples and in four of six DS-TL; no mutation was detected in 13 children with acute lymphoblastic leukemia (DS-ALL), 6 with DS-AML (M0, M2, and M5), 6 with DS-MDS and in 8 DS infants without hematological disorders and 2 children with AML M7 without DS. Blast cells proportion in the sample represented a critical aspect on the sensitivity of mutation detection in GATA1, and a combination of sequence analysis and PAGE is necessary to detect mutations when blast percentage is low. The absence of detected mutations in any of the DS-MDS cases raises the question whether MDS in DS children is an intermediate stage between TL and AML M7, as previously suggested.

Case-Control Studies↗
PubMed2000

Activation of the AT1 angiotensin receptor is dependent on adjacent apolar residues in the carboxyl terminus of the third cytoplasmic loop.

The C-terminal region of the third intracellular loop of the AT(1) angiotensin receptor (AT(1)-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G(q/11)-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232-240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile(238) or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile(238) with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe(239) with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile(238) and Phe(239) mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile(238) and Phe(239) as the critical residues in the C-terminal region of the third intracellular loop of the AT(1)-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.

Angiotensin II↗
PubMed2006

Functional interaction of CP2 with GATA-1 in the regulation of erythroid promoters.

We observed that binding sites for the ubiquitously expressed transcription factor CP2 were present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), we demonstrated the functional importance of the adjacent CP2/GATA-1 sites. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impaired HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 were present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes.

Amino Acid Motifs↗
PubMed1989

Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed.

Amino Acid Sequence↗
PubMed1993

Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region.

The malignant transformation potential of high-risk human papillomaviruses (HPVs) is closely linked to the expression of the viral E6 and E7 genes. To elucidate the molecular mechanisms resulting in HPV oncogene expression, a systematic analysis of the cis-regulatory elements within the HPV type 18 (HPV18) upstream regulatory region (URR) which regulate the activity of the E6/E7 promoter was performed. As the functional behavior of a given cis-regulatory element can be strongly influenced by the overall composition of a transcriptional control region, individual elements were inactivated by site-directed mutagenesis in the physiological context of the complete HPV18 URR. Subsequently, the effects of these mutations on the activity of the E6/E7 promoter were assessed by transient transfection assays. We found that the transcriptional stimulation of the E6/E7 promoter largely depends on the integrity of cis-regulatory elements bound by AP1, SP1, and in certain epithelial cells, KRF-1. In contrast to previous reports by implying a key role for NF1 and Oct-1 recognition motifs in the stimulation of papillomavirus oncogene expression, the inactivation of these elements in the context of the HPV18 URR did not strongly affect the transcriptional activity of the E6/E7 promoter. Mutation of a promoter-proximal glucocorticoid response element completely abolished dexamethasone inducibility of the HPV18 E6/E7 promoter and resulted in an increase of its basal activity. Functional dissection of the HPV18 constitutive enhancer region indicates that its transcriptional activity is largely generated by functional synergism between a centrally located AP1 module and thus far undetected cis-active elements present in the 5' flank of the enhancer. Furthermore, comparative analyses using homologous and heterologous promoters show that the transcriptional activity of HPV18 enhancer elements is influenced by the nature of the test promoter in a cell-type-specific manner.

Base Sequence↗
PubMed2007

Weak definition of IKBKAP exon 20 leads to aberrant splicing in familial dysautonomia.

Splicing mutations that lead to devastating genetic diseases are often located in nonconserved or weakly conserved sequences that normally do not affect splicing. Thus, the underlying reason for the splicing defect is not immediately obvious. An example of this phenomenon is observed in the neurodevelopmental disease familial dysautonomia (FD), which is caused by a single-base change in the 5' splice site (5'ss) of intron 20 in the IKBKAP gene (c.2204+6T>C). This mutation, which is in the sixth position of the intron and results in exon 20 skipping, has no phenotype in many other introns. To determine why the position 6 mutation causes aberrant splicing only in certain cases, we first used an in silico approach to identify potential sequences involved in exon 20 skipping. Computational analyses of the exon 20 5'ss itself predicted that this nine-nucleotide splicing signal, even when it contains the T>C mutation, is not sufficiently weak to explain the FD phenotype. However, the computational analysis predicted that both the upstream 3' splice site (3'ss) and exon 20 contain weak splicing signals, indicating that the FD 5'ss, together with the surrounding splicing signals, are not adequate for defining exon 20. These in silico predictions were corroborated using IKBKAP minigenes in a new rapid and simple in vitro coupled RNA polymerase (RNAP) II transcription/splicing assay. Finally, the weak splicing signals that flank the T>C mutation were validated as the underlying cause of familial dysautonomia in vivo using transient transfection assays. Together, our study demonstrates the general utility of combining in silico data with an in vitro RNAP II transcription/splicing system for rapidly identifying critical sequences that underlie the numerous splicing diseases caused by otherwise silent mutations.

Alternative Splicing↗
PubMed1995

Calpain inhibitor I decreases beta A4 secretion from human embryonal kidney cells expressing beta-amyloid precursor protein carrying the APP670/671 double mutation.

We have investigated the effects of the cell-penetrating cysteine protease inhibitors calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal) and calpain inhibitor II (N-acetyl-Leu-Leu-methioninal) on the secretion of the beta-amyloid peptide (beta A4) using transiently transfected cells expressing beta-amyloid precursor protein (APP) with the NL670/671 double mutation. Calpain inhibitor I markedly reduced the amounts of immunoprecipitable beta A4 and p3 peptide released into the culture medium. Within the cells C-terminal APP fragments accumulated. Since beta A4 secretion by cells expressing the 100 amino acid long APP C-terminus was also reduced by calpain inhibitor I, we conclude that this substance directly or indirectly interferes with the gamma-secretase activity responsible for generating the beta A4 and p3 C-termini.

Amino Acid Sequence↗
PubMed1999

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site.

Experimental probing of the model of the muscarinic M2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [3H] N-methylscopolamine and [3H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [3H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (< 10-fold) to the binding site of the M2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M2 receptor, as predicted by the proposed model.

Amino Acid Sequence↗
PubMed1998

Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

We report an extensive Li-Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT-PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.

Adult↗
PubMed1996

An enhancer LEF-1/TCF-1 site is essential for insertion site-independent transgene expression in thymus.

Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.

Aminohydrolases↗
PubMed2006

Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides.

Phosphoinositides (PIP(n)s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X(2) channels also are regulated by PIP(n)s, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X(2) channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X(2) cytoplasmic domain to anionic lipids using PIP(n)s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP(2)s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X(2) with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIP(n)s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X(2) cytoplasmic C-terminal domain.

Adenosine Triphosphate↗