2016 Lucian Award
Fifty years after it was established through a generous bequest to McGill University,1 the 2016 Louis and Artur Lucian Award for research in circulatory disease has been presented to Brian Kobilka, MD, of Stanford University. Kobilka is being honored for his enormous contributions to the understanding of G protein–coupled receptors (GPCRs), the versatile family of receptors that mediate the majority of cellular responses to hormones, neurotransmitters, and some types of sensory input.
“Kobilka revealed the molecular configuration of the β2-adrenergic receptor (β2AR) through the use of crystallography, then doggedly pursued research in this area until he discovered how the receptor functions. He has now expanded his work to a whole range of GPCRs. Many of the other candidates for the 2016 Lucian award were extremely productive and highly cited, but his science was head and shoulders above the others. With a very long array of papers in high-impact and difficult-to-publish-in journals—Cell, Science, Nature, and PNAS—Kobilka’s contributions have been recognized by his peers as cutting-edge work that advances the field significantly, not as small incremental discoveries,” says James Martin, MD, chair of the McGill University Department of Medicine and new chair of the Lucian award committee.
During his internship at Barnes Hospital, Kobilka often treated unstable patients with medications acting on GPCRs, including the adrenergic and muscarinic receptors regulating heart rate and blood pressure and opioid receptors controlling pain. His interest in intensive care medicine led Kobilka to apply for cardiology fellowships, and he was drawn to the program at Duke University because it encouraged fellows to pursue basic research. Kobilka joined the laboratory of Robert Lefkowitz, MD, where the β2AR had been identified and Jeff Benovic, a graduate student, had recently succeeded in purifying enough β2AR from hamster lung tissue to obtain several peptide sequences. Despite having far less research experience than others in the laboratory, Kobilka asked if he could contribute to the effort to clone the β2AR, a collaborative project between the laboratory and Merck.
“As a medical student and undergraduate I had done descriptive projects, but this was an exciting and challenging problem that wouldn’t respond to a cookbook approach. It would require a lot of work and some element of innovation, but obtaining the first real view of what the protein looked like could be transformative,” says Kobilka.
Brian Kobilka, MD, received his BS in biology and chemistry from the University of Minnesota Duluth and his MD from Yale University. After completing his residency in internal medicine at Barnes Jewish Hospital in St Louis, Missouri, Kobilka pursued specialty training in cardiology at Duke University in a program that encouraged fellows to pursue basic research. While working in the laboratory of Robert Lefkowitz, MD, he was part of the team that cloned the gene for the β2-adrenergic receptor, finding its sequence to be strikingly similar to rhodopsin, a seemingly unrelated receptor. This research led to the recognition of the 7 transmembrane structure shared by rhodopsin and a large number of other G protein–coupled receptors.
In 1989, Kobilka established an independent laboratory in the newly created Department of Molecular and Cellular Physiology at Stanford University, directed by neurobiologist Richard Tsien, PhD. Kobilka is now professor of molecular and cellular physiology. Between 1989 and 2003, he was an investigator of the Howard Hughes Medical Institute.
Kobilka was named a member of the National Academy of Sciences in 2011. In 2012, Kobilka and Lefkowitz were jointly awarded the Nobel prize in chemistry for their studies of G protein–coupled receptors.
Kobilka maintains an active laboratory with 10 postdocs and 2 graduate students. Kobilka met his future wife, Tong Sun Thian, when they were undergraduate students. Trained as a microbiologist and physician, Tong Sun Kobilka has worked closely with her husband for >30 years.
In weeklong excursions, Kobilka learned the basics of molecular biology from Richard Dixon, PhD, at Merck, returning to help set up the needed facilities to prepare and screen cDNA libraries within the Lefkowitz laboratory. After months of failures and false-positive hits that turned out to represent nonspecific binding with the probes, they switched approaches. Eventually, Kobilka generated a library of genomic sequences from the hamster lung DNA, which was successfully screened with a few long guessed probes at Duke and Merck.
“Usually you would only go to the genomic library as a last resort because of technical challenges, but we were very lucky that the β2AR gene was intronless so we could get the entire coding sequence from a single fragment of the gene,” explains Kobilka.
When analyzing the newly isolated gene coding for the β2AR, they discovered that the receptor was similar to rhodopsin, the light-sensitive receptor in rod cells of the retina.2 This work provided the first insight into the 7-transmembrane structure that would turn out to be a general feature of GPCR receptors.
“Throughout the time that Brian was in my laboratory, in the mid 1980s, he displayed a remarkable ability to successfully perform experiments which other people were not able to do. This wasn’t simply some great technical facility with his hands. He would devise clever, even ingenious, approaches, which others had not thought of, or even thought had no chance of working. This same ability has been manifest repeatedly over the years in his own independent laboratory program,” says Robert Lefkowitz, MD, the James B. Duke professor of medicine at the Duke University School of Medicine and a Howard Hughes Medical Institute investigator.
Crystallizing the β2AR
Cloning of the β2AR raised new questions for Kobilka: What parts of this protein were important for binding? Exactly where did the ligand bind? How did it activate the G proteins?
“Starting as a physician, I hadn’t thought much about protein structure before my time in the Lefkowitz laboratory. However, after obtaining the clone and getting the first low-resolution look at the β2AR, I wanted to determine its 3-dimensional structure, and learn how structural changes following agonist binding led to G protein activation,” says Kobilka.
After moving to Stanford, Kobilka pursued multiple lines of exploration to learn more about the function of GPCRs, including pharmacological studies and creating strains of knockout mice for several ARs to assign their roles in cardiovascular function and behavior.
Always in his mind, however, was the goal of crystallizing the β2AR to reveal how the receptor worked in molecular detail. Determining the structure of β2AR was likely to be far more difficult than it had been for the rhodopsin receptor, a remarkable achievement made easier because the protein has a rich natural source in the eyes of cows and is much more stable than other GPCRs.
“The abundance and stability of rhodopsin made it unique, and it was necessary for us to produce large quantities of β2AR, to purify it and keep it in a functional state,” Kobilka says.
After comparing different expression systems, by 1993, Kobilka was able to express and purify large enough quantities of functional β2AR in insect cells to investigate its structure with fluorescence spectroscopy—gaining important insights into the dynamic character of the β2AR that would guide their approaches to crystallizing the receptor. For several years, there were many failures and incremental improvements in strategy, and during that time, Kobilka did not enlist the help of his students and postdocs because the structural studies were too high risk to be suitable for their training. That changed, however, when he obtained crystals of the β2AR in 2004. Although the crystals were tiny, with weak diffraction, Kobilka felt that the long-sought goal was not impossible, and he could now involve 2 willing and talented postdoctoral fellows in the quest. Eventually, the team and collaborators used protein engineering to replace a particularly flexible region of the receptor with T4 lysozyme, a highly crystallizable soluble protein. In 2007, they published the first high-resolution pictures of β2AR.3
“I think everybody found it surprising that somebody like Brian, with no formal training in structural biology or x-ray crystallography, would invest himself so deeply in the remarkably difficult task of obtaining a crystal structure of the β2AR. He essentially bet his career on this, spending at least 15 years on the task and putting his funding at risk. But in the end, as we all know, he succeeded brilliantly,” says Lefkowitz.
Snapshot of a Receptor in Action
Having obtained the crystal structure of the β2AR in its inactive state, Kobilka set out to better understand the mechanism by which agonist binding to the β2AR leads to G protein activation. Over the next 5 years, he and his collaborators gained the expertise and materials that would allow the β2AR–G protein complex to be crystallized. The resulting structure of the β2AR–G protein complex4 revealed the mechanism of signal transduction across the plasma membrane, where small changes in the agonist binding pocket of the receptor propagate to large changes in the structure of the G protein (Figure).
Expansion to Other GPCRs
After all the progress in understanding the β2AR, Kobilka’s laboratory began to ask how much of what they had learned would apply to other members of the large GPCR family. In 2013, the Kobilka laboratory reported the crystalline structure of the agonist-bound active state of the human muscarinic acetylcholine receptor M2, which is in the same subfamily as β2AR and plays a key role in modulating cardiac function. They found that when bound to iperoxo, the M2 receptor—which is particularly prone to allosteric modulation—exhibited far more pronounced changes in the orthostatic binding site in the extracellular vestibule than the active states of either β2AR or rhodopsin.5
Kobilka next focused on the more distantly related μ opioid receptor (μOR) because it is the target for important pain medications and drugs of abuse, mediating both their analgesic properties (through G protein signaling) and potentially lethal side effects (through the β-arrestin pathways). The laboratory’s x-ray crystal structure of murine μOR, bound to a morphine agonist and a camelid antibody fragment, provided a model for how the differential efficacy of morphinan ligands may be encoded within small chemical differences in otherwise structurally similar molecules.6
“Brian’s findings on the structure and dynamics of the β2AR and other GPCRs have gone the furthest into finding the atomic resolution detail of how the receptors are activated. While no changes in cardiovascular medicine have as yet resulted from these fundamental findings, it would not be surprising if ultimately such changes emerge,” Lefkowitz said.
Although not in cardiovascular medicine, the promise of structure-based drug discovery involving a GPCR became reality in August with the identification of a potential analgesic using the μOR crystal structure.7 Researchers from several laboratories, including Kobilka’s, started with 3 million potential molecules and used computational docking to analyze their interactions with the μOR crystal structure. Eventually, the field was narrowed to one experimental opioid analgesic compound, PZM21, which they found to be a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Tested in mice, the compound induced longer-lasting pain relief than morphine, without causing constipation or depressing respiration.
In addition to crystallography, the Kobilka laboratory now deploys fluorescence, nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and single molecule fluorescence spectroscopy to gain insight into the dynamic properties of GPCRs.
“Having crystal structures helps us to design experiments to examine protein dynamics—the time-dependent structural changes that probably play an important role in how proteins signal. We’re trying to dig deeper into both receptor dynamics and receptor interactions with other proteins—G proteins, kinases, and arrestins,” Kobilka says.
The next deadline for a dean or department chair to nominate a scientist for the Lucian Award is March 24, 2017. Further information is available at http://www.mcgill.ca/lucianaward/
The opinions expressed in News & Views are not necessarily those of the editors or of the American Heart Association.
- © 2016 American Heart Association, Inc.
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