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Researchers get precise picture of cell target for drugs

Brian Kobilka

BY MITZI BAKER

More than half of all drugs given to patients work by targeting a particular type of "docking station," or receptor, found on body cells, to steer the cell's machinery toward healing an illness. Researchers from the School of Medicine and the Scripps Research Institute have determined what one of those receptors looks like at the molecular level, giving them the keys to greater control of the process.

A scientific feat, identifying the structure of these docking stations—called G protein-coupled receptors—can direct the future design of drugs that will bind to specific receptors. Precise binding by a drug can stimulate or block that particular receptor's normal activity, leading to more powerful treatment while minimizing bothersome side effects.

"The majority of hormones and neurotransmitters work through one of these receptors," said Brian Kobilka, MD, senior author of three publications devoted to the structure of a particular G protein-coupled receptor called beta 2-adrenergic receptor. "All these receptors are structurally related, which means that knowing more about a specific one will advance the whole field."

Kobilka, professor of molecular and cellular physiology, headed the Stanford team that published the findings with Scripps researchers in two Science Express articles on Oct. 25, showing a high-resolution structure of the beta 2-adrenergic receptor.

These publications follow on the heels of an advance online publication in Nature on Oct. 21 by Kobilka's group, demonstrating the structure of the receptor at a lower resolution but without the modifications required to see the structure at high resolution.

With more than 300 members, the G protein-coupled receptors constitute the largest family of proteins found in the membranes of cells. These cellular receptors respond to biological communication molecules—hormones, neurotransmitters and cytokines, to name a few—and function like molecular switches to promote or stifle a multitude of biological processes within the cells. G protein-coupled receptors play a critical role in heart disease, blood pressure regulation, inflammation and psychological disorders.

"These receptors are ideal candidates for therapeutics for many types of diseases," said Kobilka. He became interested in G protein-coupled receptors when he was a resident in internal medicine, witnessing how medications affecting these receptors could have a dramatic impact on his patients.

But one of the problems is that a drug that latches on to one of these receptors might also attach to a closely related one, which can trigger unwanted side effects.

"The impact of a high-resolution structure of these molecules could be really tremendous," said Kobilka. "The more you know about a receptor's structure, the more you will be able to design better drugs that are more effective and more selective."

The newly identified structure is the first high-resolution picture of a human G-protein-coupled receptor, so named for their ability to bind to G proteins, or guanine nucleotide-binding proteins. These proteins turn on and off by capturing and releasing a phosphate molecule.

Before this new structure was worked out, the only receptor for which scientists had high-resolution information was the visual sensory protein rhodopsin, from cows. Rhodopsin is highly specialized to be very sensitive to the detection of light.

The vast majority of these G protein-coupled receptors respond to hormones and neurotransmitters. They are not naturally abundant and tend to be structurally unstable.

Kobilka's lab has more than 15 years of experience in enhancing the properties of G protein-coupled receptors to make them amenable to being "seen," in a high-resolution image produced by X-ray crystallography.

Kobilka and his team decided to tackle the beta 2-adrenergic receptor, which plays a role in regulating cardiovascular and pulmonary function. Drugs targeting this receptor are used in the treatment of asthma and preterm labor.

The team faced two obstacles: obtaining enough receptor material to study, and preserving its folded shape, which is critical to its function. Daniel Rosenbaum, PhD, a postdoctoral scholar in Kobilka's lab, came up with an innovation: He replaced part of the protein with another molecule called T4 lysozyme to promote crystallization and protect the receptor from degradation while still retaining its function.

"We went out on a limb and engineered a major modification to the beta 2-adrenergic receptor," said Rosenbaum, who is one of the two first authors of the publications.

But still, the quality of the crystals was not adequate for structure determination.

At the Scripps Research Institute, Raymond Stevens, PhD, professor of molecular biology and of chemistry, and Vadim Cherezov, PhD, a scientific associate in Stevens' lab, had been working for years to develop a completely automated method of crystallizing a receptor. It involves embedding a protein in a fatty layer, much like the membrane in which it naturally resides.

Combining the techniques enabled them to see the beta 2-adrenergic receptor at high resolution.

"I am extremely happy that the technique proved itself remarkably," said Cherezov, who is the other first author of the articles. A human G protein-coupled receptor is "literally Mount Everest of membrane structural biology," he said, and seeing it in such rich detail is tantamount to reaching its summit.

This work was supported by the National Institutes of Health, the Lundbeck Foundation and the Mather Charitable Foundations.

Stanford researchers who contributed to the Science Express papers are: postdoctoral scholars Soren Rasmussen, PhD, and Tong Sun Kobilka, PhD; life science technician Foon Sun Thian; life science research associate Hee-Jung Choi; and William Weis, PhD, professor of structural biology and of molecular and cellular physiology.