Environmental Factor, August 2007, National Institute of Environmental Health Sciences
Cancer Researcher Discusses MRP1 and GSH-dependent Transport
By Robin Arnette
The 2007-2008 NIEHS Distinguished Lecture Series opened with a seminar by Susan Cole, Ph.D. (http://qcri.queensu.ca/runtime.php?NavigatorId=-111458&SiteId=-1430&ItemId=173&op=moreInfo) , on September 11 in Rodbell Auditorium. Cole, a Canada Research Chair and Queen's University Bracken Chair in Genetics and Molecular Medicine at Queen's University Cancer Research Institute, presented "The Complex Role of GSH in the Function of the MRP1 Drug and Organic Anion Transporter." The seminar was hosted by David Miller, Ph.D., head of the Intracelluar Regulation Group in the NIEHS Laboratory of Pharmacology and Chemistry.
Cole is interested in studying how drugs, toxins and metabolites get into and out of cells. The protein she cloned along with colleague Roger Deeley 15 years ago - multidrug resistance protein (MRP1) - plays a major role in conferring drug resistance in cancer patients as well as protecting normal tissues from cytotoxic drugs. MRP1 is a member of the ATP-binding cassette (ABC) transporter subfamily C and is the model for what researchers know about drug and xenobiotic efflux.
But when Cole first discovered MRP1, she had no idea how complex it was going to be. "There are 49 human ABC transporters now, but when I got started in the field there were just two known human transporters," she said. "I feel sorry for people who got into the field 10 years later because there were about 35 [transporters] then, and it made the field much more difficult because there was so much we didn't know."
One of the first things transport scientists wanted to know about MRP1 was what substrates it carried across cell membranes. Cole explained that a German research group found that MRP1 had high affinity for and could transport leukotriene C4 (LTC4 ), a pro-inflammatory glutathione (GSH)-conjugated arachidonic acid derivative. Another group knocked out MRP1 in mice and demonstrated that the mice had impaired inflammatory responses, which established that LTC4 was a physiological substrate for MRP1. "This identification made us and others start to wonder that it wasn't just this endogenous GSH-conjugated metabolite that could be a substrate for MRP1, but probably other GSH-conjugates as well," Cole said.
Other studies established that MRP1 could transport unmodified drugs, but only in the presence of GSH, so Cole and her group designed experiments that would determine the exact mechanism of action. Because estrone sulfate labeled with tritium was the most convenient GSH-dependent substrate to work with, they used it in their initial detailed mechanistic studies of MRP1-mediated GSH-dependent transport. Cole cautioned, however, that if they had used another GSH-dependent substrate, it probably wouldn't have produced the same results. The data they have generated to date is likely to be, to some degree, substrate-specific, although significant similarities are expected with other GSH-dependent MRP1 substrates.
Cole's group also performed extensive site-directed mutagenesis studies to determine which amino acids may be important for substrate binding and transport by MRP1. Their 350 point mutations of the 1531 amino acid-long MRP1 revealed phenotypes where mutant MRP1 proteins are not expressed, show global or substrate selective loss of substrate binding and/or transport activity, exhibit altered nucleotide binding and hydrolysis properties, or manifest a combination of these phenotypes.
Although Cole and her group have a better understanding of how GSH affects the transport properties of MRP1 than they did 15 years ago, there is more to learn. By continuing to look for experimental answers to their questions, they intend to fully understand the complex mechanisms that determine the ability of MRP1 to transport an extraordinarily diverse range of molecules across the plasma membrane.
Model of MRP1 GSH-stimulated Transport of Estrone Sulfate
Results from radioligand equilibrium binding assays and other biochemical studies led Cole's group to develop the following model for GSH-stimulated transport of estrone sulfate. GSH binds to MRP1 on the inside of the cell and causes a conformational change in the transporter that opens up a high affinity site for estrone sulfate. This action allows efficient binding.
Then, when ATP binds, additional conformational changes allow estrone sulfate to be transported over to a low affinity site on the outer side of the membrane so the substrate can be released; however, the inner GSH binding site remains in a high affinity state and consequently, GSH is not transported. It is only released from the membrane after ATP hydrolysis when the binding site returns to a low affinity state. "Although incomplete, this is our current explanation of how binding of both these substrates to MRP1 occurs and can affect each other's binding, but one is transported and the other isn't," Cole stated.