Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorutu electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptorrich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-'HI( -)physostigmine, the physostigmine-binding site is located within the same subunit (a polypeptide) of the receptor as the acetylcholine-binding site. Using a variety of proteolytic cleavage conditions for the purified a polypeptide, several ['Hlphysostigmine-labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand-binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and di.rect channel blockers.(-)Physostigmine (eserine), the major alkaloid of the calabar bean Physostigma venenosum Balfour [l], is a slowly reversible inhibitor of acetylcholine esterase (AChE), acting by carbamoylation of the active serine residue within the 'esteratic site' of the enzyme [2, 31. This ancient drug [4] continues to be widely used as a diagnostic aid, as an analgesic, in the recovery from anaesthesia, in the treatment of some types of psychosis, for the treatment of alcohol intoxication and glaucoma, and, most recently, for the treatment of acute brain hypoxia IS] and Alzheimer's disease [6, 71. Since physostigmine readily penetrates the bloodhrain barrier, it mainly acts in the braidcentral nervous system. In spite of its wide-spread medical applications and long use in research, however, many of its actions are not yet thoroughly understood, including those at cholinergic and serotonergic synapses [8-141. Evidence has been assembled over the past few years suggesting that, in addition to acting as an inhibitor of AChE, the drug directly interacts with both muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors [ 10-141.Studying frog muscle nAChR, we have recently demonstrated that physostigmine, below 1 pM, induces single-channel currents with amplitudes and open-time characteristics typical for the nAChR channel [12,. At higher con- [20, 211. Surprisingly, the direct action of physostigmine at the Torpedo nAChR was unaffected by the presence of saturating concentrations of acetylcholine antagonists, including a bungarotoxin and D-tubocurarine 1201, or when nAChR was desensitized by elevated concentrations of acetylcholine [21]. In addition, we established that two monoclonal antibodies raised against nAChR could competitively inhibit the binding of physostigmi...

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The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3‘-arylazido-8-azidoadenosine 5‘-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of cross-linking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.