Shedding new light on vision at the molecular level

Paper of the MonthDecember 7, 2016

Story by Rebecca Hood

November's featured paper, "Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state" in PNAS is published by Christopher Schafer, Ph.D., Jonathan Fay, Ph.D., and Jay Janz, Ph.D., a team led by David Farrens, Ph.D., associate professor of biochemistry and molecular biology, OHSU School of Medicine.

While research forays usually begin with a driving question, this project actually began with several disparate, unexpected observations made by current and former graduate students in the Farrens lab. Ultimately, they were able to piece together these puzzling findings to elucidate new aspects of how the visual receptor rhodopsin behaves.

It started when Chris Schafer (now Dr. Schafer at UT Health Science Center at Houston) was following up on some earlier work done by a former graduate student, Dr. Jay Janz  (now at Pfizer) that had gone unpublished for years. While following up on those earlier findings, Chris made, according to Dr. Farrens, "some remarkable observations that made us realize we were onto something pretty cool." The paper describing these results reveals novel insights into how rhodopsin works.

Rhodopsin, retinal, and research

Rhodopsin is categorized as a G protein-coupled receptor (GPCR), a large family of signaling receptors that are major pharmaceutical drug targets. While GPCRs have a multitude of functions, all share a common structure that spans the cell membrane (seven times, to be exact), and most begin their signaling when a molecule specific to the receptor, called a ligand, binds to and "turns on" the receptor. 

However, rhodopsin is different than most GPCRs. It starts out with its ligand, retinal, already covalently attached inside the receptor. In the dark, the bound retinal (called 11-cis retinal, or 11CR) keeps the receptor in a non-signaling inactive or "off" state. Rhodopsin only begins signaling when light converts the bound 11CR into an active agonist form (called all-trans retinal, or ATR). This induces and stabilizes the receptor in an active form that that then binds and activates downstream targets, ultimately resulting in vision.  

It was previously thought that after ATR formed inside rhodopsin, over time the linkage holding it inside the protein broke, resulting in the release of ATR and an empty receptor that was unable to signal. 

However, as Dr. Farrens explained, "We discovered that, in contrast to most prior assumptions, the release of ATR from rhodopsin is not necessarily an irreversible process. To our surprise, our data suggested that after being released, the ATR could rebind to any empty rhodopsins that had an active-like conformation."

To further test this idea, they enlisted the help of another former graduate student, Dr. Jon Fay (now at UNC Chapel Hill), who had developed and used classical pharmacological methods to study ligand binding to the cannabinoid receptor, CB1, a "traditional" GPCR. 

Dr. Farrens and his team next realized that if ATR can rebind to a receptor after leaving it, then the empty receptor must be able to exist in an active-like state after the ATR was released, i.e. without the activating ATR being bound. As he put it, "Using some novel fluorescence methods, we found compelling evidence that after the ATR is released the newly empty receptor can briefly persist in an active-like structure before ultimately collapsing to an inactive form."

Seeing GPCRs in a new light

These findings add yet another level of intricacy to the current understanding of how GPCRs work. As Mary Heinricher, Ph.D., associate dean for basic research, OHSU School of Medicine, described it, "GPCRs used to be so simple – but not anymore! And this is just a great example of the subtlety and complexity of these receptors."

Beyond elucidating the mechanism through which ATR and rhodopsin interact, these findings are notable in that they may change the way scientists consider rhodopsin and other GPCRs. According to Dr. Farrens, "These insights show rhodopsin behaves much more like the large family of GPCRs that bind diffusible drugs (opiod, cannabinoid, adrenergic receptors, etc.) than we previously thought. If the activating ATR agonist is not stably attached to rhodopsin (as has been previously assumed), but instead is constantly binding and releasing, then it should be possible to outcompete ATR rebinding with new drugs, just as is done for other GPCRs." 

In addition, the discovery that rhodopsin can exist in an active-like state after the release of ATR is novel and could have wider implications in how all GPCR activity is considered.

Looking ahead

For now, though, Dr. Farrens and his team want to further investigate the interactions between ATR and rhodopsin. 

"Our next goal is to determine when and under what conditions ATR equilibrium binding occurs to rhodopsin, to help future drug development efforts," he said. He also noted, "To me, our findings are a perfect example of why graduate students are so critically important to research at institutions like OHSU. I really don't think this discovery and work would have been achieved without the kind of interest, time and curiosity that (thankfully) graduate students bring to a research question."



Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):11961-11966. Epub  2016 Oct 4. Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state. Schafer CT1, Fay JF1, Janz JM1, Farrens DL2.

Photo: Dr. Farrens (left), Dr. Schafer

About the OHSU School of Medicine Paper of the Month

The OHSU School of Medicine spotlights a recently published faculty research paper each month. The goals are to describe to the public the exceptional research happening at OHSU as well as inform our faculty of the innovative work underway across the school’s departments, institutes and disciplines. The monthly paper is selected by Associate Dean for Basic Research Mary Heinricher, Ph.D. Learn more