How do we see? While this seems like a simple question with a seemingly obvious answer, how exactly a photon of light can trigger neural signals in our brains has been eluding biologists for decades. However, one of Cornell’s research groups, under Prof. Richard Cerione, pharmacology and chemical biology, was able to elucidate the mechanism of seeing in a recent paper published in Molecular Cell.
For several years, the Cerione Lab had been observing and experimenting on a system called phototransduction, the process by which light can activate signals in our brains and allow us to understand what we are seeing.
This process was already known to involve four components: a special kind of receptor in the eye called rhodopsin, a G-protein called transducin and two nucleotides, guanosine triphosphate (GTP) and guanosine diphosphate (GDP).
According to Sekar Ramachandran, a senior research associate in the Cerione Lab, previous research indicated that rhodopsin is activated by light, causing it to bind to and activate the transducin. However, the mechanism for this activation, as well as many details about the physical form of the rhodopsin-transducin complex form remained in the dark.
Modeling the rhodopsin-transducin complex and its activation mechanism is what the Cerione lab sought to shed light on.
The entire process begins with rhodopsin, a member of a large family of receptors called G-protein-coupled receptors (GPCRs). Rhodopsin has several special properties, most notably, it can be activated by a single photon of light. Once it is activated, a change in its physical form allows it to bind to transducin and form a rhodopsin-transducin complex.
Transducin is a special kind of protein called a G-protein. G-proteins bind certain nucleotides. In this case, unactivated transducin is bound to GDP. However, the formation of the rhodopsin-transducin complex causes the transducin to activate, releasing its GDP and binding instead to GTP.
This binding causes the transducin molecule to break away from the rhodopsin and continue the signal to the brain. Afterwards, the remaining activated rhodopsin can continue to activate previously-unactivated transducin at a rapid pace. “You amplify the signal by [10,000] times [in this way],” said Yang Gao, A postdoctoral researcher.
While trying to characterize the receptor-G-protein complexes, three main problems arose: first, the group wanted a three-dimensional model of the complex in its active state, with the GTP attached to the transducin. However, since the binding of GTP is inherently unstable, it was difficult to keep the structure from breaking apart.“They have to turn over very quickly,” Gao said, “So it’s difficult to hold them together long enough for you to do experiments with them.”
The second difficulty is isolating the complex outside of its natural environment, as this introduces additional instability.
“[The transducin] will stay on with high affinity on the membrane [of the cell], but when they’re on the membrane you can’t study it, because the membrane is hard to manipulate,” Gao said. “You have to extract it out of the membrane, so we spent a long time trying to figure out how to get it out of the membrane and keep it stable.”
The lab managed to remedy this issue by utilizing nanobodies, a special kind of protein created naturally in immunized llamas that binds to the complex and prevents it from breaking apart.
However, the use of nanobodies creates a hurdle that few researchers have managed to overcome — including a Nobel Prize winner.
To illustrate the caveat of working with nanobodies, Ramachandran described the work of Robert Lefkowitz and Brian Kolbilka on another GPCR called the beta-2 adrenergic receptor, for which the pair received the 2012 Nobel Prize in Chemistry.
Although the pair of Nobel Laureates, like the Cerione Lab, were ultimately able to form a three-dimensional model of the receptor, slight alterations in the physical form of the complex due to nanobody use prevented the exact replication of the natural complex.
However, the resulting three-dimensional model they obtained has proven helpful in answering several questions concerning the complex.
Gao relates that transducin is formed of three subunits, named alpha, beta and gamma. The alpha subunit is shaped like a clamshell, with its “jaws” being called the helical domain and the Ras domain. When the transducin is inactive, the domains hold the GDP inside. When the GPCR binds to the transducin, the domains separate, releasing the GDP and allowing GTP to attach.
Until now, the mechanism by which the receptor opens the clamshell was a mystery, and scientists assumed that the beta and gamma subunits probably served some role in holding the two domains apart. However, Cerione Lab was able to specifically identify the role that the beta subunit plays in this action.
According to Gao’s model, the beta subunit acts like a doorstop by attaching itself to the helical domain, and introducing certain mutations that would weaken the “glue” between the beta subunit and helical domain causes a significant decrease in signaling speed, indicating that activated transducin with weaker “glue” took longer to replace its GDP with GTP.
GPCRs are vitally important parts of many biological processes. As much as 34 percent of FDA-approved drugs target them. As a result, GPCR research remains an area of intense research.