December 1, 1995 - On the Quad

On the quad:

MOLECULAR MIDDLEMEN




































Eva Neer


Of all the chemical signals knocking against the surface of the cell, few have greater impact than the hormones. A minuscule amount of a messenger hormone like glucagon or epinephrine can bring about tremendous changes in the body-raising blood sugar or hastening the beating of the heart.

While other hormones such as estrogen deliver their messages directly into the cell, hormones such as glucagon and epinephrine must pass their messages on, via hormone receptors, to a group of molecular go-betweens lying in wait beneath the membrane of the cell.

These molecular middlemen, known as G proteins, signal other molecules, such as enzymes or ion channels, which, in turn, trigger a whole series of changes inside the cell.

For more than 20 years, Eva Neer, Harvard Medical School professor of medicine, has been trying to understand how G proteins help hormones get their messages into cells. Her research, in collaboration with scientists at HMS and elsewhere, is revealing a new side to this once mysterious set of molecules.

For years, it was believed that G proteins activate enzymes and ion channels through the alpha subunit, one of two main components in a G protein. But several years ago, Neer and her collaborators showed that the other main component of a G protein, the beta-gamma subunit, also plays an activating role.

Amplifying the Message

The beta-gamma subunit is turning out to be surprisingly versatile and enterprising. In addition to relaying messages from the hormone receptors to enzymes and ion channels, it appears to act as a multiple-docking site for proteins. This multiple-docking capability could enable a hormonal message to be dispersed through several chemical pathways, thereby amplifying the message.

Neer first came at the G proteins through the molecular "back door." Her original interest, back in the early 1970s, was in an enzyme at the far end of the hormone-receptor-G- protein-enzyme relay system, adenylate cyclase. Adenylate cyclase was known to be triggered by epinephrine, glucagon and other hormones. But at the time that Neer began her work, little was known about what adenylate cyclase looked like or how it worked.

One reason adenylate cyclase was difficult to get a handle on was that it was caught in a delicate place-inside the cell membrane. "If one can think of such a benighted time, people weren't very clear whether membrane proteins were different from other proteins. It was just sort of the beginning of trying to figure out what were the properties of proteins in membranes as opposed to proteins in solution," says Neer.

Using the methods of the day-traditional biochemical approaches-Neer isolated the protein and took it apart to identify its parts. She then became interested in how adenylate cyclase was turned on and off. At the time, adenylate cyclase was known to be regulated by a particular kind of protein, one that appeared to bind to the molecule GTP (guanosine triphosphate). This protein also appeared to turn receptors on and off. "It became clear that there was this middleman protein between the receptor and the cyclase enzyme," Neer says.

The middleman was a G protein, so dubbed because it binds to GTP. G proteins were found to have some striking peculiarities. To begin with, they appear to be quite choosy: they respond mostly to hormone receptors that snake through the membrane seven times, no more and no less.

It also became clear that G proteins bind to GTP only when activated. In their inactive state, they bind to a related molecule, GDP (guanosine diphosphate). When activated by a hormone receptor, the G protein changes its shape, shaking GDP off. GTP then swoops in to take its place, propelling the G protein to interact with an enzyme or an ion channel. At some point, the GTP molecule is cleaved into GDP, which returns the G protein to an inactive state.

The G proteins were also found to be made up of three polypeptide units-alpha, beta and gamma. The latter two were welded together to form a tight unit, the beta-gamma subunit. Most researchers-including Neer-believed that it was the alpha subunit that played the active role, binding GTP and activating adenylate cyclase. The beta-gamma subunit was thought to play primarily a stabilizing role.





















Inactive G proteins bind GDP (left). Once activated by a hormone receptor R (center), GDP is replaced by GTP (right). The alpha and the beta-gamma subunits separate. Both subunits may activate an enzyme E.


But then, in 1984, Neer and her colleagues discovered another G protein, this time in the brain. In the course of investigating this new protein, Neer and her colleagues David Clapham and Diomedes Logothetis tried to see whether the protein's alpha subunit could open a potassium ion channel. To their surprise, it couldn't.

"We said, 'Well, put on the other one [the beta-gamma subunit].' And sure enough, it activated very nicely-which of course was different from the prevailing viewpoint," Neer says. The beta-gamma subunit in fact plays an active role in several G-protein mediated interactions. Recently researchers have discovered that the beta subunit in particular consists of a series of seven joined petals. "It's this absolutely gorgeous molecule," Neer says.

The beta subunit is a member of a large-and ancient-family of proteins, known as WD proteins. These "petaled" proteins are found in all eukaryotes-from slime molds to humans. And they are remarkably unchanged in each, despite hundreds of millions-even billions-of years of evolution.

Protein Terminal

"There's something that's commonly good [about all of the WD proteins]," Neer says. She speculates that one function of the individual petals is to create specific places for proteins to bind. At the same time, the multipetaled structure of the WD proteins may provide an opportunity for several proteins to dock at once-much like an airline terminal. "It creates a surface for protein-protein interactions," Neer says.

This new understanding of G proteins could help explain how hormonal messenger molecules can have such profound effects on the body. By joining together several different proteins, a G protein would spread the hormonal word down several chemical pathways, thereby amplifying the original message.

Neer is currently investigating where and how the receptors and effectors bind to G proteins in vitro. She is also exploring how G proteins work in living animals. She and her colleagues are inserting defective (overly active) G protein genes into mice to see what effect that has on them. They are also planning to knock out the G protein gene in mice.

Eventually, Neer's work could lead to new treatments for a wide variety of genetic diseases associated with G protein deficiencies. Most important of all, it could lead to new treatments for heart disease and growth problems related to defects in the hormonal relay system.

"If you want to find specific ways to modify particular pathways, you must understand the complexity of the pathway," Neer says. "The more one knows about how the signal gets across-and particularly what keeps it specific-[the more] you might be able to design better drugs. But that's a very long way off."

--Misia Landau