One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner.

We describe a rapid and effective procedure for purifying recombinant eukaryotic TATA binding protein (TBP) from Escherichia coli. The method employs an affinity ligand comprising glutathione-S-transferase fused to the carboxyl-terminal activation domain of the transcriptional activator VP16 and an amino-terminal domain (TAND2) of the yeast TBP-associated factor TAF1. TBP can be purified without the need for extrinsic affinity tags, subsequent proteolysis, or downstream clean-up steps. This TBP purification process is rapid (requiring about 4h after bacterial harvest) and does not require sophisticated chromatographic equipment. The resulting material is monodisperse, structured, and functionally active. We demonstrate the efficacy of this method for purifying recombinant full-length or TBP core fragments encoded by yeast, humans and Arabidopsis.

A unique phenotype of 5-HT2C, agonist-induced GTPgamma35S binding, transferable to 5-HT2A and 5-HT2B, upon swapping intracellular regions.

1 The human 5-HT(2C) receptor, when expressed heterologously in various mammalian cell lines (HEK293, SH-EP and NIH-3T3) at various receptor densities (6 to 45 pmol mg(-1) protein), mediates robust agonist-induced GTPgamma(35)S binding from coupling to G(i) subtypes of G proteins, in addition to G(q/11). Such a phenotype, however, was not seen with the human 5-HT(2A) and 5-HT(2B) receptors, indicating their common pathway with 5-HT(2C) limited to G(q/11), not including G(i). 2 Because intracellular regions are largely responsible for signalling pathways, we prepared the chimeras of the 5-HT(2A) and 5-HT(2B) receptors where the second and third intracellular loops, and the C-terminal region were replaced with the 5-HT(2C) counterparts. 3 The chimeras showed robust agonist-induced GTPgamma(35)S binding. Relative intrinsic efficacies of agonists from the GTPgamma(35)S binding were nearly identical to the reported values for their parent receptors as measured with Ca(2+) or [(3)H]-inositol phosphate accumulation. Also the chimeras displayed the same ligand-binding properties as the parent receptors. 4 We conclude that the phenotype of agonist-induced GTPgamma(35)S binding is unique to 5-HT(2C) among the 5-HT(2) receptor family, and is transferable to 5-HT(2A) and 5-HT(2B), upon swapping intracellular sequences, without altering their receptor pharmacology.

Discovery of a protein sequence in the N-terminal region of the human neuronal alpha7 nicotinic acetylcholine receptor involved in homomeric interactions.

The neuronal nicotinic acetylcholine receptor subunit, alpha7, can form homopentameric receptor/ion channel complexes. Potential contributions of its N-terminal region to homomeric interactions were investigated, in comparison with the corresponding region of an analogous heteromeric subunit, alpha3. Recombinant chimeras were prepared upon engineering the N-terminal alpha7 (M1-V224) or alpha3 (M1-S232) sequence into the background of another homomeric mouse 5-hydroxytryptamine3 (5-HT)(3) receptor. The alpha7/5-HT(3) chimera, when expressed heterologously in a human epithelial cell line, SH-EP1, robustly expressed alpha-bungarotoxin binding sites as homooligomers while the alpha3/5-HT(3) did not produce epibatidine (non-selective ligand) binding sites, and did not interfere the alpha7/5-HT3 phenotype, upon co-expression. Yeast two hybrid assays with the N-terminal regions showed positive responses between alpha7:alpha7, but not between alpha7:alpha3 and alpha3:alpha3. Similar assays with the alpha7 N-terminal region and its five smaller fragments (G23-N46, D47-N90, V91-N133, S134-M182and Q183-V224) revealed that the G23-N46 sequence is involved in homomeric interactions. Replacement of the corresponding region of the alpha3/5-HT(3) chimera with the alpha7 G23-N46 sequence conferred a dominant negative role on the chimera, by abolishing the alpha7/5-HT(3) phenotype. These results support the view that the G23-N46 portion of the alpha7 N-terminal region may contribute to receptor homooligomerizations.

Positive allosteric modulator of the human 5-HT2C receptor.

The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.