The underlying architecture of a cellular signalling complex involved n the body's response to stimuli such as light and pain has been determined by a team of scientists from Duke Medicine, the University of Michigan and Stanford University.
This complex, consisting of a human cell surface receptor and its regulatory protein, reveals a two-step mechanism that has been hypothesized previously but not directly documented.
The findings, reported on June 22, 2014, in the journal Nature, provide structural images of a G-protein coupled receptor (GPCR) in action.
Lefkowitz is co-senior author with Georgios Skiniotis, Ph.D., the Jack E. Dixon Collegiate Professor at the Life Sciences Institute at the University of Michigan, and Brian K. Kobilka, M.D., the Helene Irwin Fagan Chair in Cardiology at Stanford University School of Medicine. Lefkowitz and Kobilka shared the 2012 Nobel Prize in Chemistry for their discoveries involving GPCRs.
GPCRs represent the largest family of drug targets for human diseases, including cardiovascular disorders, neurological ailments and various types of cancer. The protein beta arrestin is key for regulating these receptors, and the authors have visualized a complex of the protein beta arrestin along with the receptor involved in the "fight-or-flight" response in humans.
"Arrestin's primary role is to put the cap on GPCR signaling. Elucidating the structure of this complex is crucial for understanding how the receptors are desensitized in order to prevent aberrant signaling," Skiniotis said.
"High-resolution visualization of this signaling assembly is challenging because the protein complexes are transient and highly dynamic and large amounts of the isolated proteins are required for the experiments," said co-lead author Arun K. Shukla, who worked with Lefkowitz at Duke and is now setting up an independent laboratory in the Department of Biological Sciences and Bioengineering at the Indian Institute of Technology, Kanpur.
Once the authors had material available for direct structural visualization, they used electron microscopy to reveal how the individual molecules of this signaling assembly are organized with respect to each other.
The researchers then combined thousands of individual images to generate a better picture of the molecular architecture. They further clarified this picture by cross-linking analysis and mass spectrometry measurements.
The authors next aim to obtain greater detail about this assembly using X-ray crystallography, a technology that should reveal atomic level insights into this architecture. Such atomic details could then be used in experiments to design novel drugs and develop a better understanding of fundamental concepts in GPCR biology.
"This is just a start and there is a long way to go," Shukla said. "We have to visualize similar complexes of other GPCRs to develop a comprehensive understanding of this family of receptors."
In addition to Lefkowitz, Shukla, Skiniotis and Kobilka, study authors include Gerwin H. Westfield; Kunhong Xiao; Rosana I. Reis; Li-Yin Huang; Prachi Tripathi-Shukla; Jiang Qian; Sheng Li; Adi Blanc; Austin N. Oleskie; Anne M. Dosey; Min Su; Cui-Rong Liang; Ling-Ling Gu; Jin-Ming Shan; Xin Chen; Rachel Hanna; Minjung Choi; Xiao Jie Yao; Bjoern U. Klink; Alem W. Kahsai; Sachdev S. Sidhu; Shohei Koide; Pawel A. Penczek; Anthony A. Kossiakoff; and Virgil L. Woods Jr.
Howard Hughes Medical Institute provided funding, along with the National Institutes of Health (DK090165, NS028471, GM072688, GM087519, HL075443, HL16037 and HL70631); the Mathers Foundation; the Pew Scholars Program in Biomedical Sciences; the Canadian Institutes of Health Research; and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES.