Cells and signals

Americans Robert Lefkowitz and Brian Kobilka win the Chemistry Nobel for piecing together the puzzle of cells responses to the environment.

THE human body consists of billions of specialised cells which work through a fine-tuned network of interactions among them. For example, the cells in the eye, the nose and the mouth have sensors for light, odours, tastes and flavours respectively. How do these cells pick up signals for these sensations from the outside world? How does the body sense the outside environment and adapt to new situations? We have known since the late 19th century that the central nervous system acts as the master control system and sends appropriate signalschemicals such as hormones and neurotransmittersfor the various kinds of cells to respond to a given situation, including extreme threats that trigger a fight-or-flight response in us. The glands in the body begin to pump out larger quantities of hormones such as adrenalin (medical name epinephrine) into the blood stream, the heart begins to pump faster, and fat cells, muscle cells and lungs begin to respond appropriately and immediately. How do the insides of these cells (cytoplasm) communicate with the bodys mobile messengers that are outside? This had remained a mystery for a very long time.

It turns out that the cells use a set of proteins called G-protein-coupled-receptors (GPCRs), natures most versatile biological sensors, to which external signalling molecules bind much like a key fitting into a lock. This years Nobel Prize in Chemistry has been awarded to Robert Lefkowitz, a 79-year-old professor of biochemistry from Duke University Medical Centre, North Carolina, United States, and Brian Kobilka, a 67-year-old former student of Lefkowitz and currently a professor of molecular and cellular physiology at the Stanford University School of Medicine, California. The award is for unravelling the inner workings of this important family of GPCRs in the 1980s. Today, the knowledge provided by the discoveries of Lefkowitz and Kobilka is part of medical school textbooks and it is taken for granted that GPCRs are proteins on cell surfaces that mediate cellular responses to hormones, neurotransmitters, odourants, chemokines and taste by flitting in and out of the cell surface seven times.

Scientists had for long suspected that the cell surface must contain some sort of a recipient for hormones, but the exact nature of these and how they worked had remained obscure for most of the 20th century. By the mid-19th century, it was known that every human cell is covered by a plasma membrane comprising two layers of phospholipids (fat molecules). The membrane enables the cells to maintain the correct balance of biochemically active species within it while stopping the entry of unwanted substances from the outside. The trigger that sets these biochemicals into action is a signal from the outside sent out by the brain in the form of chemicals such as hormones.

For example, it was known that when adrenalin is administered to the outside of a cell, there is a change in its metabolism that can be measured inside the cell. The question then was: how did the signal get through the wall? How could the inside of the cell have known the changes happening outside? Although rhodopsin was identified as far back as the 1870s as the photosensitive pigment in the retina, the ligand (a molecule that binds with a biomolecule to enable a biological function) known as retinal that binds to rhodopsin was discovered in 1933, and the process of vision was fully understood by the late 1950s, it was not known that rhodopsin was a GPCR. This became clear only after Lefkowitz and Kobilka unravelled the generic structure of a GPCR.

What is interesting is that despite the lack of a detailed understanding of this cellular communication, drugs, whose effect depend critically on the binding of drug molecules to these receptors, have been developed. In the 1940s, the American scientist Raymond Ahlquist had empirically concluded that there must be two types of receptors for adrenalin: one that chiefly makes smooth muscle cells in blood vessels contract and another that stimulates the heart. He called them alpha and beta. This led to the development of beta-blockers, which still constitute some of the essential medicines for the heart. But after a couple of decades, five years after Lefkowitz began his research career in 1968, Ahlquist wrote about the adrenergic receptors that he had postulated: They were an abstract concept conceived to explain observed responses of tissues by chemicals of various structures. Even Edwin Sutherland, who won the Nobel Prize in 1971 for the discovery of adenylate cyclase, a key regulatory enzyme found in nearly all cells, had posited that the beta receptor and adenylate cyclase are the same.

Enter Lefkowitz with his ambition to become a cardiologist. Having graduated at the height of the Vietnam War, he did his military service in the U.S. Public Health Service at the National Institutes of Health (NIH) where his supervisor, Jesse Roth, had already a plan to find the receptors by tagging hormones with radioactive iodine. He set Lefkowitz on the job. Both Roth and Ira Pastan, his colleague at the NIH, had realised that to validate the existence of receptors, it would be important to develop a biologically relevant direct ligand-binding assay. Radioactively labelled assay seemed appropriate. The idea being that as the hormone bound itself to the cell surface, the receptor could be tracked from the radiation emitted by iodine. Lefkowitz was thrown an additional challenge: to show that the coupling to the cell surface actually triggered the biochemical process inside the cell.

Lefkowitz began his work with adrenocorticotropic hormone, which stimulates the production of adrenalin in the adrenal gland, and studied its binding to adrenal membrane preparations. However, even after a year, nothing seemed to work. Lefkowitz finally made progress as the project entered its second year. In 1970, he published two papers in the prestigious journals Proceedings of the National Academy of Sciences (PNAS) and Science on the discovery of an active cell-surface receptor. The thrill of making a discovery hooked him on to research for good and he moved to Duke University.

Cardiology continued to interest him, and at Duke he began work on receptors for adrenalin and noradrenalin, hormones that regulate many different physiological processes, the heart rate and blood pressure in particular. Using radioactively labelled substances, including beta-blockers, his research team, after a great deal of effort, extracted a series of adrenergic receptors, which included one of Lefkowitzs main goals, the beta receptors. Following that discovery, the field literally exploded with specific radioligands used to identify a large number of receptors.

Meanwhile, knowledge about the biochemical processes inside the cell had been growing with the discovery that G-proteins are activated by a signal from the receptor (Nobel Prize for Physiology or Medicine 1994). The G-proteins, in turn, set off a cascade of reactions that alters the metabolism of the cell. By the 1980s, an understanding of the process by which signals are transmitted from the outside of the cell to the inside began to emerge and key to this was the so-called ternary complex model put forward by Lefkowitz and his associates (Figure 1). The first step towards this was Lefkowitzs idea to look for a gene that coded for the beta receptor. He felt that if his group could isolate the blueprint for the beta receptor coded in the gene, one would be able to decipher how the receptor worked.

At about the same time, Lefkowitz recruited into his group a young doctor by name Brian Kobilka. Kobilka was fascinated by adrenergic receptors from his experience in hospital intensive care where a shot of epinephrine could mean life to a dying patient. In 1984, wanting to study the role of epinephrine in its molecular detail, Kobilka had come to join Lefkowitzs laboratory. Soon Kobilka joined the hunt for the gene.

However, this was not an easy task as the receptor was produced in small quantities. The team, with all the receptor that could be collected, managed to determine only a few parts of its genetic sequence. It was not the modern days of genomics when the full genome sequence is readily available for the appropriate gene to be identified and cloned. Kobilka then struck upon an ingenious idea, the first of many flashes of technological innovation, as Lefkowitz says. Kobilka built a library of mammalian genomic sequences with a plan to screen it with the scraps of sequences that the group had. This would enable the team to extract longer sequences which, when stitched together, would reveal the full gene sequence. The plan succeeded because of the fortuitous circumstance that the gene had no introns (unimportant nucleotide sequences within a gene), and pieces could be just stitched together.

The stitched sequence showed seven long and fatty (hydrophobic) helical strings of amino acids that are typically found in cell membranes. This suggested that the receptor probably moved in and out of the cell wall seven times. This also rang a bell because this was just like the rhodopsin, which activated retinal, also a G-protein. There was no a priori reason for both to be similar: one was activated by a hormone and the other by light. It was a real eureka moment, recalls Lefkowitz. About 30 proteins were then known to activate different G-proteins. That gave rise to a thought of deep significance: there has to be a complete family of receptors that look alike and function the same way. (Interestingly, for isolation and purification of these water-insoluble beta adrenergic receptors, Lefkowitz had used the same detergent as used earlier in the isolation of functional rhodopsin even though the sequence homology between the two was not known then.)

Since this path-breaking discovery, the pieces of the puzzle of transmission of signals into a cell slowly began to fall in place. As a result of follow-up work by several scientists, we now have a detailed knowledge of how GPCRs work in general and how they are regulated at the molecular level. Although signals picked up by GPCRs range from photons and odourants to hormones and neurotransmitters, signal transmission is accomplished by a highly similar process by a family of structurally related receptors with seven trans-membrane helices. An important aspect of this mechanism is that the hormone or any other ligand does not pass through the membrane. Instead, the signal is transferred to the inside of the cell by changes in the shape of the GPCR that couples to the ligand on the outside. The higher the concentration of the ligand, the greater the fraction of receptors binding to the ligand. The receptor in the membrane is dynamic and can assume different shapes. Such changes in structural arrangements set off a cascade of reactions on the intracellular side beginning with the binding of the G-protein to the receptor (Figure 2). A small conformational change at one end of the receptor gets amplified by the helical framework into a much larger conformational change at the other intracellular end. One can imagine the receptor as a bundle of seven rod-like structures immersed in the membrane. When the ligand grips the bundle at one end, it opens up as a bouquet of flowers at the other, which enables the G-protein to bind. This essentially constitutes transmission of the signal from the outside to the inside of the cell (Figure 3).

After the successful isolation of the receptor gene, Kobilka moved to Stanford University School of Medicine. There he set out to image the receptor, which, in the opinion of many scientists, was almost impossible to do. But Kobilka had set his mind on it and it was to be a two-decade-long journey. In 2011, he and his team of researchers reported their success: a high-resolution image of the receptor at the very moment when it transfers the signal from the hormone on the outside to the G-protein on the inside. This was certainly a crowning achievement.

Imaging a protein, which is too small to be seen through regular microscopes, is a challenging process involving many steps. Scientists use the method of X-ray crystallography for which you need to crystallise the proteins to begin with. When X-rays are directed through the crystal, its tightly packed atomic centres scatter the radiation, leaving behind a diffraction pattern that tells how the atoms are arranged in the protein.

The first image of a protein by X-ray crystallography was done in the 1950s. Since then, thousands of proteins have been imaged. However, most of them were soluble in water, which enables easy crystallisation. Just as oil, GPCRs lodged in fatty membranes of the cell are water-insoluble and they tend to form fatty lumps. Moreover, they are produced in very small quantities. But more significantly, given their function, GPCRs are mobile, but for crystallisation they have to be steady. Since it is the membrane that holds them in shape, getting them out intact and stable can be most frustrating. Crystallising GPCRs, therefore, poses a great challenge, and nobody had attempted that before.

The imaging of the rhodopsin structure by the Polish biochemist Krzysztof Palczewski in 2000, too, was not of much help to Kobilka because rhodopsin can be isolated in large quantities from animal eyes, which can be collected in large numbers from slaughterhouses. Also, rhodopsin is simpler and more stable than other GPCRs. To compound his frustration, in 2003, his main funding from the Howard Hughes Medical Institute was stopped. But his irrational optimism, as one of his colleagues described it, kept him going even though his laboratory began to struggle financially. Even then he never considered giving up, according to a 2011 report in Nature soon after his success. I enjoyed the challenge and I wanted to know the answer, he had said. Finally, in 2004, his group succeeded in growing tiny crystals of beta receptors but too small to be analysed with the X-rays from Stanfords synchrotron facility. He then took his samples to the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. But the crystals diffracted only to a poor resolution of 20 ngstroms () against the 2 or 3 required to distinguish the individual atoms in the crystal (1 is a tenth of a billionth of a metre, 10-10m).

In 2005, there was good news on funding from the NIH. But the problems with growing bigger crystals continued to be frustrating, especially because of a constantly moving and floppy intracellular part. Finally, the group hit upon the idea of anchoring the loose end in place by attaching an antibody to it. With this technique, his postdoctoral researcher Soren Rasmussen succeeded in growing bigger crystals. The first measurements with these crystals gave an exciting result with a 3.5 resolution. The structure published in Nature in 2007 was the second crystal structure of a GPCR after rhodopsin. Soon after, with another postdoctoral researcher, Vadim Cherezov, Kobilka used a new technique to stabilise the beta receptor for crystallisation. Kobilka and associates published back-to-back papers in Science in the same year that determined the beta receptor structure to 2.8 . This trio of papers by Kobilka and company marks a milestone in structural biology.

Kobilka was set on finishing the GPCR story. The structures determined hitherto were GPCRs in an inactive state. For a complete understanding, the receptor needed to be imaged in the act of being activated by a ligand and turning on the G-protein. This was an even more formidable task. With help from different experts, and after relentless testing of thousands of crystallising conditions, Kobilka and Rasmussen succeeded in stabilising and holding the receptor with the G-protein through a support system involving a combination of suitable detergent, a lipid scaffold and an antibody that could hold it together.

The resulting crystal was bigger than anything that he had produced before. This time the team used the X-ray beam line of the Advanced Photon Source at Argonne National Lab, Illinois, U.S., one of the worlds brightest synchrotron-based X-ray sources, because here the beam could be more tightly focussed than at the ESRF to yield a better resolution.

Today, with the mapping of the human genome, about a thousand genes that code for GPCRs have been identified. About half of these receptors are part of the olfactory system. A third of them are responsible for hormones and neurotransmitters, such as dopamine, serotonin, prostaglandin and histamine. Others are responsible for sight and taste. There are about a hundred receptors whose roles are still to be deciphered. In fact, Lefkowitz and colleagues found in the late 1990s that GPCRs bound not only to G-proteins on the intracellular side but also to other proteins called arrestins. Because of this, GPCRs are now increasingly being referred to as the seven trans-membrane (7TM) receptors.

From a pharmacological perspective, knowledge about these receptors would be of immense benefit to humankind. About half of todays drugs, which include beta-blockers, anti-histamines and medications relating to nervous disorders, target nearly 800 human GPCRs. However, as Kobilka said in the post-Prize announcement interview, We dont really know how to control [these medicines] very well yet. [They] arent still perfect. They have side-effects. So we still have a long way to go before we can really take advantage of what we know about them in terms of therapeutics.

For Kobilka, the task is far from over. He is already working to understand what the various active states of the receptor look like, why different GPCRs couple to different G-proteins, and what happens when different ligands bind to the same receptor, which is not uncommon. He is also using diverse techniques, such as electron microscopy and nuclear magnetic resonance, to understand the flexibility of GPCRs. If you are really interested in something enough, Kobilka said in his interview, you just keep working on it.

Brian ultimately reaches his goals, says Lefkowitz in Nature. Sometimes it takes 15 years, but he gets there.