HOX A genes are induced in a temporal fashion after retinoic acid (RA) treatment in non-N-ras-transformed PA-1 human teratcarcinoma cells. However, In N-ras-transformed PA-1 cells, RA-Induced expression of HOX A genes is delayed. The mRNA for the transcriptional activator AP-2 is overexpressed in these ras-transformed cells, but AP-2 transcriptional activity is inhibited relative to non ras-transformed PA-1 cells. Constitutive expression of AP-2 mimics the effect of ras by transforming cells and inhibiting differentiation in culture. We analyzed 4 kb of the human HOX A4 gene promoter and identified seven putative AP-2-binding sites in the DNA sequence. Transcription assays with variably sized HOX A4 promoter reporter constructs revealed that a 365 bp region of the promoter, -2950 to -3315 relative to the mRNA start, controls RA responsiveness and ras-mediated inhibition of HOX A4 activity. This region contains an AP-2 binding site and a RARE. Elimination of the AP-2 site by site-directed mutagenesis demonstrated that the AP-2 site is involved in RA-mediated transcriptional activation of the human HOX A4 promoter in combination with the RA receptor response element (RARE). In N-ras-transformed cells, low HOX A4 promoter activity results from ras inhibition of AP-2 transactivation.

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.

Retinoic acid (RA), a developmental morphogen, causes activation of a transcript of an endogenous retrovirus-related element in the human teratocarcinoma-derived cell line PA-1. This provirus is defective, and the provirus-related sequences exist as multicopy elements (more than 20 copies) in human DNA. This is the first human endogenous retroviral mRNA that is known to be transcriptionally activated by RA. The nucleotide sequence of the 3,357 bp of this viral cDNA was determined and shows a strong homology to the type C-related human endogenous retroviral proviruses ERV3 and 4-1. This cDNA contains 'R-U5-Apol-env-U3-R sequences of the provirus. Adjacent to the putative 5' long terminal repeat of this provirus there is an 18-bp sequence complementary to the 3' end of isoleucine tRNA. We named this RA-responsive virus RRHERV-I.

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