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Single-molecule techniques illuminate mechanisms of GPCR activation

Findings from scientists at St. Jude Children’s Research Hospital and Columbia University/New York State Psychiatric Institute reveal how GPCR signaling is regulated with implications for drug development.

Memphis, Tennessee, April 27, 2022

St. Jude researchers Daniel Terry and Scott Blanchard

Daniel Terry, Ph.D., and Scott Blanchard, Ph.D. of the Department of Structural Biology

Scientists at St. Jude Children’s Research Hospital and Columbia University/New York State Psychiatric Institute are studying G protein-coupled receptors (GPCRs), membrane proteins that are the target of one-third of approved drugs. Using single-molecule imaging techniques, researchers gained fresh insight into the process by which cellular signals are relayed by GPCRs. The work may aid the development of novel drugs by manipulating the way they activate certain pathways. A paper on the work appeared today in Cell.

The human genome encodes roughly 800 GPCRs, which are expressed throughout the human body and control many physiological functions. GPCRs are important drug targets for a wide variety of disorders.

GPCRs transmit signals inside the cell when activated by agonists, (which are chemical signals such as neurotransmitters, hormones, cytokines or synthetic drugs). β-arrestins are proteins that bind GPCRs to stop G protein-mediated signaling and can also trigger a variety of other downstream signaling pathways.

The interaction of a GPCR with β-arrestin is mediated in part by a specific region of the GPCR, typically its tail. This tail undergoes activation-dependent phosphorylation. The phosphorylated GPCR tail docks into a groove on β-arrestin. But when β-arrestin is not engaged with a GPCR, this groove is occupied by β-arrestin’s own C-terminal tail. Researchers wanted to understand how the β-arrestin C-terminal tail gets released to make room for the phosphorylated GPCR tail.

“We’re trying to understand the process by which information is transmitted from the outside to the inside of the cell through GPCRs and, specifically, whether this information transfer occurs by way of detectable conformational [shape] changes in GPCRs and their binding partners,” said co-corresponding author Scott Blanchard, Ph.D., St. Jude Department of Structural Biology. “Single-molecule imaging is a way to directly measure molecular-scale conformational changes that is very insightful and often more easily interpreted than other approaches.”

A tale of two tails

Using single-molecule fluorescence resonance energy transfer (smFRET), the researchers monitored the conformational dynamics (shape changes) of the β-arrestin C-terminal tail. Most work in this area has been done with ensemble methods that average hundreds of thousands of proteins. These averages can’t show what a specific protein is doing. In contrast, the single-molecule approach can provide direct evidence about an individual protein’s behavior and allow the researchers to study how it changes over time.

The group’s findings show that the resting β-arrestin exists normally in a stable, autoinhibited state. In this state, the C-terminal tail is tightly bound to the groove. In order for the C-terminal tail to release and make room for the phosphorylated GPCR tail, an agonist must bind to the GPCR, which in turn “tickles” the β-arrestin to trigger the release. smFRET allowed the researchers to tease out the contributions of GPCR's phosphorylated state from its agonist-activated state, properties that can’t be separated and studied in the cell. The ability to discern these states led to the discovery that the receptor tail itself also plays an autoinhibitory role that must be relieved by agonist binding.

The balance between the autoinhibited and activated states controls the intensity and duration of GPCR signaling, which in turn determines physiological responses. The findings may play a role in drug development as they allow for a systematic exploration of the outcomes from the pattern and extent of GPCR phosphorylation.

“Now that we know receptors can both activate G-proteins and mediate signaling through β-arrestin, the hope is that the field can develop more specific pharmacotherapies by finding small molecules that preferentially activate one pathway or the other,” said co-corresponding author Jonathan Javitch, M.D., Ph.D., Columbia University and the New York State Psychiatric Institute.

Authors and funding

The study’s co-first authors are Daniel Terry, St. Jude; Wesley Asher, Columbia University and New York State Psychiatric Institute; and G. Glenn Gregorio, Weill Cornell Medicine. Other authors on the study are Alem Kahsai, Li-Yin Huang, and Robert Lefkowitz, Duke University Medical Center; Bing Xie and Lei Shi, National Institute on Drug Abuse National Institutes of Health; Ying Zhu, Columbia University and New York State Psychiatric Institute; Wonjo Jang and Nevin Lambert, Medical College of Georgia, Augusta University; Alekhya Govindaraju, New York State Psychiatric Institute and University of California Berkeley; Asuka Inoue, Tohoku University; Vsevolod Gurevich, Vanderbilt University; and Alessandro Borgia and Arnab Modak of St. Jude.

The study was supported by grants from the National Institutes of Health (R21NS102694, R01MH054137, R01HL016037, R35GM122491, and R01GM130142); the Hope for Depression Research Foundation; the Brain and Behavior Research Foundation NARSAD Young Investigator Award; the National Institute on Drug Abuse Intramural Research Program; the PhRMA Foundation; and ALSAC, the fundraising and awareness organization of St. Jude.


St. Jude Children's Research Hospital

St. Jude Children's Research Hospital is leading the way the world understands, treats and cures childhood cancer, sickle cell disease, and other life-threatening disorders. It is the only National Cancer Institute-designated Comprehensive Cancer Center devoted solely to children. Treatments developed at St. Jude have helped push the overall childhood cancer survival rate from 20% to 80% since the hospital opened more than 60 years ago. St. Jude shares the breakthroughs it makes to help doctors and researchers at local hospitals and cancer centers around the world improve the quality of treatment and care for even more children. To learn more, visit, read St. Jude Progress, a digital magazine, and follow St. Jude on social media at @stjuderesearch.