OCT1 Function Research Articles

Polymorphisms affecting function of the human organic cation transporter hOCT1 (SLC22A1): what are the consequences?

It is well established that polymorphic genetic variations in some drug-metabolizing enzymes can dramatically affect the handling of certain drugs in the body, sometimes with severe consequences for toxicity and therapeutic efficacy [1]. More recently, polymorphisms in the active drug efflux transporter MDR1 P-glycoprotein have also been associated with changes in drug disposition, although the effects observed to date are fairly modest [2]. In this issue of the journal, Kerb et al. [3] potentially add a new twist to the cocktail of factors affecting drug disposition. In a Caucasian population, the authors found many polymorphisms in the human polyspecific organic cation transporter 1 (hOCT1) gene SLC22A1, some of which result in amino acid substitutions that severely reduce and alter substrate transport in in-vitro assays. The question that immediately emerges is what pharmacological parameters will be affected in vivo by such altered OCT1 function?

Silencing Mediator for Retinoid and Thyroid Hormone Receptors Interacts with Octamer Transcription Factor-1 and Acts as a Transcriptional Repressor

Octamer transcription factor-1 (Oct-1) is a member of the POU (Pit-1, Oct-1, unc-86) family of transcription factors and is involved in the transcriptional regulation of a variety of gene expressions related to cell cycle regulation, development, and hormonal signals. It has been shown that Oct-1 acts not only as a transcriptional activator but also as a transcriptional repressor for certain genes. The mechanism of the repressive function of Oct-1 has not been well understood. Here we demonstrate by using the glutathione S-transferase pull-down assays and coimmunoprecipitation assays that the POU domain of Oct-1 directly interacts with a silencing mediator for retinoid and thyroid hormone receptors (SMRT). The interaction surfaces are located in the C-terminal region of SMRT, which are different from previously described silencing domains I and II or receptor interacting domains I and II. In transient transfection assays in COS1 cells, overexpression of SMRT attenuated the augmentation of Oct-1 transcriptional activity by OBF-1/OCA-B, activator for Oct-1. In pull-down assays, increasing amounts of SMRT could compete the binding of OCA-B to Oct-1 POU domain. The activity of Oct-1 could be determined by a regulated balance between SMRT and OCA-B. Furthermore, cotransfected unliganded thyroid hormone receptor enhanced the transactivation by Oct-1, and addition of 3,3',5-tri-iodo-l-thyronine obliterated the stimulatory effects. Consequently, in the presence of cotransfected thyroid hormone receptor, the octamer response element acts as an element negatively regulated by 3,3',5-tri-iodo-l-thyronine. The results suggest that the transcriptional activity of Oct-1 can be modulated by interaction through its POU domain by a silencing mediator SMRT resulting in the cross-talk between Oct-1 and nuclear receptors.

Coactivation by OCA-B: definition of critical regions and synergism with general cofactors.

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators.

Analysis of transcriptional stimulation by recombinant Oct proteins in a cell-free system.

The transactivation potential of several isoforms of the lymphoid-specific transcription factor Oct2 has been analyzed using in vitro transcription. Oct2 can stimulate transcription in B-cell nuclear extracts and in HeLa nuclear extracts depleted of the ubiquitous factor Oct1 by wheat germ lectin affinity chromatography. Activity is observed from both natural and synthetic promoters containing single or multiple copies of the octamer motif ATGCAAAT. Multimerization of this motif does not result in a synergistic transcriptional stimulation, but rather leads to a linear increase in activity. To analyze the various Oct2 isoforms, they were overexpressed in HeLa cells using recombinant vaccinia virus. Although all the isoforms bind similarly to the octamer sequence, they show clear differences in their ability to transactivate transcription. This ranges from a 2-fold stimulation for Oct2.3 to the almost 20-fold effect of the most potent variant Oct2.5. In general the relative activity of the isoforms in vitro reflects that observed in vivo in cotransfection experiments. Interestingly the ubiquitous factor Oct1 is also an efficient activator of transcription in vitro, but only from promoters with multiple octamer motifs. Sarkosyl inhibition studies suggest that both Oct1 and Oct2 function in vitro by stabilizing preinitiation complexes without affecting the reinitiation rate of RNA polymerase II.