The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide.

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Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease.

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.The multidrug resistance protein, MRP1, 1 is believed to function as an active exporter of many conjugated organic anions from cells (1-7). Among agents that are transported are also some unconjugated compounds, including certain cancer drugs, provided that glutathione is also present (8, 9). In this case, the hydrophilic conjugating compound such as glutathione may be co-transported along with the hydrophobic drug (8). As a member of the ABC super family of membrane transporters (10), MRP1 is an ATPase that is stimulated by agents that it transports (11, 12). However, as yet there is little further information about how the two nucleotide binding domains of the protein act to bring about the transport event. In the case of the better studied P-glycoprotein, which belongs to a different family of this super family (13), the two NBDs have been shown to be functionally equivalent with identical ATP hydrolysis steps occurring alternatively at each domain (14, 15). According to an insightful model based largely on this information, binding and translocation of the hydrophobic compound exported is controlled in an ordered fashion by the two hydrolysis steps (15). The objective of the present study was to determine whether MRP1 performs a similar symmetrical cycle of partial reactions of ATP hydrolysis to accomplish the export of conjugated organic anions. To follow the interaction of the nucleotide substr...