In 1986, Brian Kobilka, of Stanford University School of Medicine, was a member of the team that cloned the ß₂-adrenergic receptor, a molecule that spans the cell membrane and that responds to the presence of adrenaline by triggering diverse components of the "fight or flight" response. More than 20 years later, he is one of the lead authors on two Top Ten papers that describe the detailed molecular structure of ß₂AR, as the receptor is known. Getting from there to here required a succession of technical and conceptual breakthroughs that have paved the way to a far greater understanding of the most common family of trans-membrane signal receptors.

There are about 1,000 G protein-coupled receptors (GPCRs) that respond to a huge range of stimuli, from the light that activates the visual pigment rhodopsin to hormones and small molecules such as adrenaline. As Kobilka and his colleagues note in the authors’ summary of one of the highly cited papers (#2), "drugs that act on GPCRs command more than 50% of the current market for human therapeutics, with annual revenues in excess of $40 billion." But those drugs often have untoward side effects; asthma drugs, for example, can make the heart beat too fast if the dose is not carefully controlled. Part of the reason is that drug design is difficult because the structure and function of the receptors are not well understood. And that is not surprising.

Rhodopsin, which was characterized around a decade ago, is unusual in being physically quite stable, which means it is relatively easy to make the crystals needed to determine its structure. ß₂AR is much "wobblier" and is in any case hard to crystallize because the surface of the molecule that sits within the membrane tends to be hydrophobic and thus to steer clear of the close molecular contacts that are essential to crystal formation. It also seems likely that ß₂AR, being a trans-membrane protein, needs to be within a membrane to exhibit its true shape.

Kobilka’s team adopted two approaches to the problem. In both, they stabilized the outside of the receptor by binding it with the beta-blocker carazolol. For the inside, one group bound one of the intracellular loops to a monoclonal antibody. The other genetically engineered the ß₂AR molecule, replacing the same intracellular loop with a small protein derived from T4 bacteriophage. The antibody and the T4 lysozyme both encouraged the formation of a crystal lattice. Of course there is more to mapping the molecular structure than just having the crystals, but without the crystals nothing is possible. To have two different sorts of crystal is fortunate indeed.

"We didn't know which [method] was going to work, so we tried both," Kobilka recently told The Scientist (23[2]: 51, 2009). "And they ended up both working at about the same time."

The paper by Rasmussen et al., at #8, reported the structure based on the monoclonal antibody and was published in Nature a week before Cherezov et al., at #2, published in Science with a higher-resolution version derived from the engineered ß₂AR. (A third paper by the team, in the same issue of Science [D.M. Rosenbaum, et al., 318(5854): 1266-73, 2007], just missed the current Top Ten with 22 citations this period.) The two structures are all but identical. Perhaps the biggest surprise is that an ionic lock, which holds the intracellular parts of the molecule together in inactivated rhodopsin (and which may be partly responsible for that molecule’s stability) is broken in ß₂AR, even though the agonist carazolol is blocking the receptor and thus might be expected to have locked the structure. If just one of the structures had demonstrated a broken lock, it might have been dismissed as an artefact, but given that it shows in both structures, even though they had different intracellular components and different lipid supports, the suggestion is that this is an important aspect of ß₂AR’s functioning.

There are other aspects of the structure that suggest ways in which the receptor actually works. For example, there is a channel through the middle of the molecule that seems to be filled with water. This could provide space for the components of the receptor to move around, reacting to different signal molecules by adopting different positions and shapes and thus possibly helping to trigger subtly different responses within the cell.

Other researchers have already made use of the ß₂AR structure, and even more so the insights that went into determining it, to pursue their own GPCRs with considerable success. Kobilka’s group is in hot pursuit of the structure of active ß₂AR, bound not by a blocker but by its correct agonist adrenaline. That may reveal details of how exactly it activates the G protein, and will probably require the crystallization of an even more complex three-part molecule—signal, receptor, and G protein responder. That is unlikely to take another 20 years.

Dr. Jeremy Cherfas is Science Writer at Bioversity International in Rome, Italy.

KEYWORDS: G PROTEIN-COUPLED RECEPTORS, GPCRS, BETA-2 ADRENORECEPTOR, BRIAN KOBILKA, BETA2 AR.