Contact: Bob Kuska
NCI Press Office
This week in the journal Science, researchers at the National Cancer Institute (NCI) report that they have an answer. In the first study to visualize the process in a living cell, the scientists found, as expected, that an activated receptor migrates to the transcription-promoting region of its target gene, setting in motion the gene-expressing machinery in the cell nucleus.
But contrary to the long-held assumption that the receptor remains fixed on the gene throughout the process, the scientists observed that it leaves as transcription gets under way. "These receptors don't stick around long," said Gordon Hager, Ph.D., an NCI scientist and senior author on the study. "It is a remarkably dynamic process in which the receptors shuttle to the gene, then shuttle off again, even in the constant presence of hormone."
This finding, based on a study of the steroid hormone glucocorticoid and its receptor, points the way to a new, more dynamic paradigm for steroid receptor function. Hager said it also raises the possibility that as steroid receptors cycle, they may be available continuously for modification by proteins from other, growth-regulating pathways. "It may be that steroid receptors have a more complex role in signaling than previously thought," he said.
According to Hager, steroid hormone receptors come in three varieties, based on where they reside in the cell when unbound by hormone. These include nuclear proteins, such as the estrogen receptor; cytoplasmic proteins, which include androgen and corticosteroid receptors; and proteins that localize in both the nucleus and cytoplasm, such as the progesterone receptor.
Researchers have learned over the years that after a hormone enters a cell, it attaches tightly to its own unique protein receptor. This interaction activates the receptor, whereupon the receptor, with the hormone riding piggyback, seeks out and binds to its target gene.
But the next steps in the process have remained murky. The prevailing theory has been, like pressing down a button on a switchboard, the receptor had to remain fixed on the gene for transcription to proceed. But this idea, presented in most biology textbooks, has come into question in recent years as studies have suggested that the receptors might shuttle to the gene momentarily and leave. This model is called the "hit-and-run" theory.
According to James McNally, Ph.D., an NCI scientist and a lead author on the paper, the speculation arose because there has been no way to directly study the receptor-gene interaction. "Traditionally, scientists have studied the question by pulling proteins out of cells and studying them in test tubes, which creates an artificial environment that yields no direct evidence about how the protein behaves in a living cell."
By the mid-1990s, as many powerful new research tools reached the laboratory, the tide began to turn. Scientists in the field, including those in Hager's laboratory, learned how to create intact steroid receptors tagged with small, green-fluorescent proteins, raising the hope that they could visually track the glow of the receptors inside of a living cell.
But, as McNally noted, this advance raised another problem. Because there are relatively few steroid receptors in a cell, those that were tagged in initial studies produced a faint fluorescent glow that was difficult to detect against the broad background of the cell. "It was a case of poor resolution," he said. "Typically, six to eight receptor molecules bind to each gene site, but that is still not enough to see very well. What was needed was a unique cellular feature that would draw out the binding activity from the background noise."
That is when luck entered into the picture. By chance, Hager's group had produced a mouse tumor cell line that spontaneously integrated a stretch of DNA that contained approximately 200 copies in a row of the transcription-promoting gene sequence for glucocorticoid. This translated to 800 to 1,200 binding sites in one place.
According to Waltraud Müller, Ph.D., an NCI scientist and a lead author on the paper, if the glucocorticoid receptors did indeed bind to this abnormally long array in the mouse tumor cells, they would have the needed resolution to observe the green-labeled receptors in action. "It seemed like this system might work, but, then again, we had no assurances that it would," said Müller.
As this week's paper reports, the system did work. Müller and her colleagues found that when the cell line was exposed to glucocorticoid, receptors for the hormone bound en masse to the array. This allowed them to ask the next questions. Do the receptors remain rooted at the binding site throughout the transcription process? Or, as some suggested, is it a case of hit and run?
To get the answer, Hager and his group conducted an experiment in which brief pulses of laser light, which were directed at the array, bleached the fluorescence from the bound receptors. Less than two seconds after the laser pulse, the scientists said they could detect other glucocorticoid receptors already binding to the array to take their place, indicating that these molecules are extremely mobile.
Then, when the laser pulse was applied elsewhere in the nucleus, Hager and colleagues discovered that the tagged receptors present on the array were rapidly replaced with untagged versions. This provided strong evidence that the receptors were coming and going from the array at a rapid rate, a finding consistent with the "hit-and-run" theory.
"What is the purpose of this rapid exchange?" asked Hager. "The receptors may come on and off in such a dynamic manner to permit continuous interactions with proteins from other signaling pathways in the cell, instead of having to wait for the removal of hormone. If so, the plot will thicken."
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