Researchers Identify Mechanism That May Regulate Protein Involved In Sex Differentiation, Stress, Hunger

If verified, the finding would signify a major breakthrough in understanding the modulation of a key regulator of the genes involved in such endocrine activities as the determination of sex, the development of sex organs and the way the body reacts to stress.

The finding could also, perhaps, be applied more generally, possibly explaining why some hormonally regulated cancers, such as of the breast and prostate, at times become resistant to anti-hormone therapies. For it suggests a possible alternative mechanism of action on these nuclear receptors in some cancer cells.

The finding, reported in the current issue of Molecular Cell, confirms what researchers have long suspected but been unable to prove - that the protein, a nuclear receptor known as steroidogenic factor 1, is regulated not by the "small molecule" hormones that regulate all other known nuclear receptors, but by a signaling cascade that culminates in phosphorylation, the addition of a phosphorus molecule to the receptor.

Nuclear receptors are a sophisticated brand of cellular proteins located inside the nucleus, where they receive signals and interact with other proteins to initiate changes in gene activity. The small-molecule hormones, or "ligands," that regulate most of these nuclear receptors sweep into a cell's nucleus from far-flung cells and latch on to their respective receptor, prompting it to bind with nearby molecular "co-factors." Together, the receptor and its co-factors bring about the change in a given gene's behavior.

The molecule, or ligand, that signals the SF-1 nuclear receptor to prompt a change in gene activity has been elusive, however, leading to its designation as an "orphan" receptor, and leaving researchers suspicious that some signal other than a small-molecule hormone might be orchestrating its actions. The UCSF study suggests this is the case.

The researchers determined that the SF-1 receptor is regulated by a cascade of signals initiated outside of the cell that, through a series of molecular events, ultimately cause a change in the molecular makeup of the receptor in the nucleus. This change, known as phosphorylation - the addition of a phosphate to the molecule - occurs in SF-1 at an activation region known as AF-1. And once it has, it prompts the receptor to recruit its co-activators, located at the traditional ligand-binding site, to help bring about changes in gene activity.

"It's been dogma that nuclear receptors are regulated by ligands," said the senior author of the study, Holly A. Ingraham, PhD, an associate professor of physiology and obstetrics, gynecology and reproductive sciences at UCSF. "Our work shows that phosphorylation of a single serine residue within an active domain (AF-1) of SF-1 modulates the activity of this nuclear receptor in the apparent absence of a ligand."

"The phosphorylation event somehow causes cross talk between the AF-1 domain being phosphorylated and the domain that interacts with the co-activators." (Similar results have been reported by Tremblay et al, in the same issue.)

The phosphorylation event itself appears to be initiated by a signaling pathway outside the cell known as map kinase.

The lead author of the study, Gary D. Hammer, MD, PhD, a research physiologist in the Departments of Physiology and Medicine at UCSF, was awarded the Endocrine Society 1999 Merck Senior Fellow award for this work last week.

The researchers conducted their investigation by trying to tease out the suspected role of phosphorylation. They did so in part by phosphorylating two versions of SF-1 - one mutated to be nonphosphorylatable, one a naturally occurring form - and observing the ability of both to activate two SF-1 target genes. One gene, MIS, is a growth factor that blocks development of the Mullerian duct, which would otherwise become the oviduct, the uterus and vagina. The other gene is P450 21 hydroxylase, a steroidogenic mouse enzyme.

The overall finding was that phosphorylation led to more recruitment of co-activators by SF-1, which in turn, led to greater activation of the gene being targeted.

The finding suggests, said Ingraham, that the mechanism would allow this nuclear orphan receptor to function much like a rheostat, elevating or diminishing gene activity. Understanding the way in which SF-1 is regulated could offer insight into how the receptor responds to stress signals and initiates the production of sex steroids. "This molecule is at the cross-roads of how endocrine signaling is achieved," said Ingraham.

The finding could also lead to a recognition of a more general ligand-independent mechanism of action in nuclear receptors in some cancers. Some hormonally induced cancers, such as of the breast and prostate, are initially sensitive to drugs that block hormone receptors, like the drug tamoxofen at the estrogen receptor. But sometimes these cancers go through stages of being resistant to hormone blockers, and it could be, said Ingraham, that the cancers are finding a way around the hormone-binding sites and signaling the receptors to produce estrogen or testosterone via another mechanism - perhaps the extracellular signal that culminates in phosphorylation.

On another level, the findings regarding SF-1 may have led researchers to a new appreciation for the complexity of nuclear receptor action and how these functions may have evolved. In recent years, scientists have determined that most nuclear receptors have phosphorylation sites, and that they play a secondary role to ligand binding. In these cases, the phosphorylation site, or AF-1 activation domain, is located at a distant location on the molecule from the ligand-binding site.

On the SF-1 receptor, in contrast, the domain being phosphorylated is directly adjacent to the ligand-binding domain, from which the co-activators function. And this, said Ingraham, indicates that even though the AF-1 domain moved locations through evolution, the function of the domain has been conserved.

"Our wildest speculation says SF-1 may have been one of the first primordial receptors, and that it received signals via phosphorylation signals, not via ligands. When the AF-1 domain was moved down to the other end of the molecule in these receptors, then the need for ligands may have been more pressing."

The researchers' next step is to try to determine at a structure level if this ligand-binding domain is reconfigured, bypassing the need for a ligand.

"Phosphorylation might well be the principal method of modulation of this orphan receptor," said Ingraham, "though we still cannot exclude the possibility that SF-1 activity depends on a ubiquitous ligand present in most cell types."

Other co-authors of the study were Irina Krylova, of the Department of Physiology at UCSF; Yixian Zhang, of the Department of Cell Biology at Baylor College of Medicine; Beatrice D. Darimont, PhD, postdoctoral fellow of the Department of Cellular and Molecular Pharmacology at UCSF; Kimberly Simpson, a UCSF Summer Student and Nancy L. Weigel, PhD, associate professor of the Department of Cell Biology at Baylor College of Medicine.

The study was funded by the National Institutes of Health.