Regulation of human P2X1 promoter activity by beta helix-loop-helix factors in smooth muscle cells.

We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.

Guanylyl cyclase stimulatory coupling to K(Ca) channels.

We coexpressed the human large-conductance, calcium-activated K (K(Ca)) channel (alpha- and beta-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell K(Ca) currents by 21 +/- 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K(Ca) currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K(Ca) currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the alpha-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate K(Ca) channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.

Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C.

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or alpha2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gbeta gamma stimulation of phosphoinositide 3-kinase gamma (PI-3Kgamma), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling.

Cloning and characterization of the promoters of the maxiK channel alpha and beta subunits.

Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences.

Fli-1b is generated by usage of differential splicing and alternative promoter.

The proto-oncogene Fli-1, a member of Ets family is rearranged or activated through proviral integration in erythroleukemias, induced by Friends' Murine Leukemia Virus. The DNA binding domain (ETS domain) of Fli-1 is fused to the RNA binding domain of EWS by t(11q24:22q12) chromosomal translocation in Ewing's sarcoma and primitive neuroectodermal tumors. Screening of human cDNA libraries has identified two different 5'-termini and alternatively spliced forms of the human Fli-1 gene (Fli-1b), suggesting the possible existence of two independent promoters. The genomic sequence adjacent to the alternate exon of human Fli-1b gene shows functional promoter activity when cloned in promoter-less CAT expression vector and transfected into QT-6 cells. The transcription initiation (CAP) site and minimum promoter region necessary for function were localized. The 5'-flanking regions of human Fli-1b and mouse Fli-1 show 80% homology suggesting conserved promoter regulatory elements. The Fli-1b 5'-flanking sequence lacks canonical TATA or CCAAT boxes but contains a partially conserved TATA-like sequence at position 242. Several transcription factor binding sequences like ATF/CREB, E2A-PBX1, EBP, PEA-3, ETS-2, Sp-1, c-Myc, TBP, GATA-1 and Oct-3 were conserved in the promoter sequence. Functional promoter assays revealed that Fli-1b promoter shows very strong transcriptional activation compared to Fli-1 promoter. We also showed that variant Fli-1b has transcriptional activation properties similar to those of Fli-1. Fli-1b and Fli-1 show differential expression in various hematopoietic cell lines. This differential expression and promoter activities of Fli-1 and Fli-1b suggests that several mechanisms are involved in Fli-1 gene regulation which are mediated by many transcription factors.

Reconstitution of beta-adrenergic modulation of large conductance, calcium-activated potassium (maxi-K) channels in Xenopus oocytes. Identification of the camp-dependent protein kinase phosphorylation site.

The human large conductance, calcium-activated potassium (maxi-K) channel (alpha and beta subunits) and beta2-adrenergic receptor genes were coexpressed in Xenopus oocytes in order to study the mechanism of beta-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 +/- 5% and 35 +/- 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the alpha and beta subunits versus those expressing the only the alpha subunit, suggesting that the alpha subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of beta2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the alpha subunit almost completely inhibited beta-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 +/- 4%). These data demonstrate that physiological coupling between beta2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the alpha subunit on the channel.

The human P2X4 receptor gene is alternatively spliced.

ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X4 gene. Clones were identified from a human stomach cDNA library using a rat P2X4 probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X4 cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5'-untranslated region and the first 90 amino acids in the coding region of full-length human P2X4 are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaperonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X4 message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT-PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X4 regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.