Cell Talk: The Players

by Neil Caudle

Ask Ken Harden to characterize his role in the full-court press of cell-signaling research at Carolina, he’ll laugh and say, "I’m the cheerleader."

But Harden, professor of pharmacology, is a player, too. Twenty years ago, he and his colleagues set out to understand how hormones regulate intracellular calcium levels. As they learned about G-protein signaling and its role in the effects of adrenaline, they hypothesized that an analogous system might be involved in the regulation of calcium. They were right. Phospholipase C (PLC), an enzyme found in the plasma membrane of all cell types, catalyzes the production of "second messengers" that affect a host of significant processes in cells.

Recently, Harden and John Sondek solved the structure of a portion of PLC-beta, one of the three classes of PLCs. In PLC-beta, the team found structural features that will help explain its function as a "GAP," a category of proteins that regulate the on-or-off decisions of G-protein-activating receptors.

"Nature," says David Siderovski, "has evolved a system we call G-protein-coupled receptors to sense what is in the cell’s environment and then communicate that to machinery in the interior of the cell. So think of a hormone that’s coursing through your veins, or a neurotransmitter that’s causing one nerve to spark another nerve, or photons of light that strike the back of your retina to give you a picture of what you see around you. All of those systems communicate into the cell some message. And the machinery that transacts that signaling is the G-protein-coupled receptor."

In this simplified diagram, serpentine receptors on the cell surface connect to a G protein composed of three parts (alpha, beta, and gamma subunits). When a signaling molecule binds to the surface, the receptor’s alpha unit exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP). This separates G-alpha from G-beta and G-gamma, opening a "switch" that activates a signaling pathway inside the cell. The switch remains open until GTP loses a phosphate through hydrolysis and reverts to GDP.

But there’s more to the story. Consider our response to light. When the lights go out, we see darkness instantly. If vision were governed by the normal rate of GTP hydrolysis, we would spend several seconds seeing the last image that reached our eyes before the lights went out. Fortunately, in this case, other proteins rush in to help clip off the extra phosphate and close the switch.

As a group, these proteins are known as "guanosine triphosphatase-activating proteins" or GAPs. Recently, Siderovski and others independently discovered a new family of GAP proteins, which they call "regulators of G-protein signaling" or RGS proteins. When it senses the activated alpha, an RGS protein can bind quickly to the "switch" regions, accelerating the shutoff. Because they apparently regulate many kinds of signaling, some or all of the 27 RGS proteins identified in the human genome are likely to become targets for various drugs.

While conventional chemotherapy attempts to kill tumor cells through a process called apoptosis, many tumor cells resist this cell-death mechanism. Al Baldwin and his colleagues have found that chemotherapy drugs can also activate the NF-kB cell-signaling pathway, which tells cells to live. Baldwin, associate professor of biology, has discovered ways to paralyze the NF-kB cell-signaling system, enhancing chemotherapy. Results so far have been promising in animal studies. The next step will be human trials including pancreatic and colon cancers, breast cancer, and myeloma.

"A tumor cell does not listen in the same way as a normal cell," says Channing Der, professor of pharmacology at the Lineberger Comprehensive Cancer Center. "So we’re trying to understand what differences in signal trans-duction occur in a normal cell versus a cancer cell, with the long-term goal of determining ways to correct that, to make the cancer cell a normal cell, or to usethe information to develop better diagnostic tools."

Der and Adrienne Cox, assistant professor of radiation oncology, are finding molecules that inhibit the RAS oncoprotein, which is mutated and apparently expressed in a wide variety of human cancers. Some of these inhibitors have shown dramatic abilities to block tumor growth in experimental models and are now being tested in clinical trials.

John Sondek, assistant professor of pharmacology, purifies a signaling protein and grows it in a crystal smaller than a grain of table salt. Then, in the ominous, bunkerlike confines of Research Building B, he bounces x rays through the crystal while a futuristic-looking machine turns it in a precisely calibrated rotation. The result, after intensive computer-aided analysis, is a three-dimensional image of the crystal structure of cellular proteins.

In December, Sondek and his colleagues published, in Nature, the crystal structures of two proteins that bind together to help trigger the spread of cancer cells. Deciphering exactly how these proteins function normally—and what can go wrong when either protein is mutated—could lead to anticancer drugs that specifically target diseased cells and leave normal cells alone.

Sondek studies the Rho family of G proteins, which normally help regulate such functions as cell shape, division, movement, and proliferation. These G proteins are also implicated in cancer, especially when under the influence of an activator known as Tiam1, whose mutated form has been shown to induce noninvasive cancer cells to metastasize—to spread and become invasive.

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