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Wind Me Up and Turn Me Loose: NIH and NIDCD-Funded Scientists Describe the Molecular Moves of Sensory Cells

From left to right, the PLCβ enzyme in closed (inactive) and open (active) positions.
From left to right, the PLCβ enzyme in closed (inactive)
and open (active) positions.
Credit: Graphic by Angeline Lyon, Ph.D.

To eat an oyster, you have to twist and pry apart its tough shell to get to the succulent mollusk inside. As it turns out, the ability to taste that briny oyster is due to a similar process of twisting and opening that goes on at the molecular level among G protein-coupled receptors and the enzymes they control.

G protein-coupled receptors isn't a phrase that easily rolls off the tongue. But GPCRs, as scientist call them, are the movers and shakers of the cellular world. They, and the other proteins they activate, are the key players in a process known as signal transduction, which is how cells relay information from outside the cell to inside the cell to make things happen. GPCRs regulate the basic pathways for the messenger molecules in the body—such as hormones and neurotransmitters—and they also control sensory pathways.

More than 800 GPCR genes in humans have been identified. A large number of these are associated with the activation of an enzyme called phospholipase Cβ (PLCβ), whose mechanism of regulation has proven elusive to researchers over the last two decades. PLCβ holds within its clamshell-like grasp a key section where the enzyme is activated and which makes it possible for us to taste, smell, see, hear, and regulate blood pressure, among many other physiological processes.

A recent finding by a group of NIH-funded researchers in the University of Michigan laboratory of John Tesmer, Ph.D., in collaboration with NIDCD intramural scientists in the cellular biology laboratory of John Northup, Ph.D., finally describes the molecular mechanism for the activation of the PLCβ enzyme. The discovery is published in the August 7 online edition of Nature Structural and Molecular Biology.

According to Dr. Northup, PLCβ proved to be an extremely "recalcitrant" enzyme. To try to peek inside it, the scientists employed a technique called X-ray crystallography. Using PLCβ purified from retinas dissected from cuttlefish and squid (which has a protein sequence similar to human PLCβ) from Dr. Northup's lab, the researchers in Dr. Tesmer's lab grew crystals from the enzyme. Each crystal contained trillions of orderly packed PLCβ molecules. They then exposed the crystals to high-powered X-rays. By studying the diffraction patterns of the crystal they were able to produce a three-dimensional image of the molecule so that it could be studied in atom-by-atom detail.

All G protein-coupled receptors act in similar ways. The pathway begins with the receptor sitting on the surface of the cell, attached to a G protein that remains in an inactive state until a signaling molecule binds to the receptor. The activated G protein splits apart to expose sections that can interact with other molecules. In this case, the G protein is specifically looking for a place to bind on the PLCβ enzyme.

It finds it in a strand of the enzyme that is floppy and flexible and a little bit lost, without the tight structure of surrounding areas. "It's flapping around continuously and rearranging its structure," says Dr. Northup. "It's basically waving around and saying 'Grab me.'"

The G protein grabs onto the strand, flips it, twists it, and stabilizes it into a new configuration. The movement is huge in terms of the size of the enzyme, spanning a distance as long as the protein itself. This massive displacement opens the enzyme like a clamshell being pried apart, allowing phospholipids (PIP2) to enter and be cleaved by the enzyme to produce the second messenger chemicals inositol triphosphate (IP3) and diacylglycerol (DAG).

A whole slew of events are now set into motion by IP3 and DAG that activate additional proteins, which activate still other proteins, in a cascade of tightly regulated and highly amplified chemical reactions inside the cell.

With this discovery, Drs. Tesmer and Northup and their colleagues are hoping to characterize even more of the protein structure of the PLCβ enzyme, since they know, judging from the amount of activity they've measured in the recently plotted section, that not all of the G protein interaction site has been found yet. "Because the section of the protein missing from the structure is so flexible and sensitive to enzymatic cleavage, it's difficult to stabilize it in crystal form" explains Dr. Tesmer.

According to the scientists, looking further down the road, knowing how this enzyme is regulated will be fundamental to understanding a diverse array of physiological processes in the body. The activity of the PLCβ enzyme affects how neurons communicate with one another and how electrical signals are transmitted in the auditory system, and it is also critical in modulating neuronal function in the development of the ear and the nervous system.

The GPCR pathways are also important in disease states—over half of the currently marketed pharmaceuticals target them—but the next generation of therapeutics is likely to focus on these downstream pathways, rather than the receptors themselves. Drugs that regulate these second messenger pathways could be used in combination with medicines that work on GPCRs to refine the therapies. Each could be used in smaller amounts and made more specific and more targeted.

"Once we learn what the contacts are that enable the mechanisms of regulation," Dr. Northup says, "we'll be able to design ways to look for things that will alter it."

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