Format

Send to

Choose Destination
Elife. 2020 Feb 25;9. pii: e54208. doi: 10.7554/eLife.54208.

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells.

Author information

1
Department of Psychiatry, University of California, San Francisco, San Francisco, United States.
2
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
3
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.
4
Center for Clinical Pharmacology, Washington University School of Medicine, St. Louis, United States.
5
St Louis College of Pharmacy, St. Louis, United States.
6
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States.
7
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States.
8
Department of Anesthesia, University of California, San Francisco, San Francisco, United States.

Abstract

G protein-coupled receptors (GPCRs) signal through allostery, and it is increasingly clear that chemically distinct agonists can produce different receptor-based effects. It has been proposed that agonists selectively promote receptors to recruit one cellular interacting partner over another, introducing allosteric 'bias' into the signaling system. However, the underlying hypothesis - that different agonists drive GPCRs to engage different cytoplasmic proteins in living cells - remains untested due to the complexity of readouts through which receptor-proximal interactions are typically inferred. We describe a cell-based assay to overcome this challenge, based on GPCR-interacting biosensors that are disconnected from endogenous transduction mechanisms. Focusing on opioid receptors, we directly demonstrate differences between biosensor recruitment produced by chemically distinct opioid ligands in living cells. We then show that selective recruitment applies to GRK2, a biologically relevant GPCR regulator, through discrete interactions of GRK2 with receptors or with G protein beta-gamma subunits which are differentially promoted by agonists.

plain-language-summary:

About a third of all drugs work by targeting a group of proteins known as G-protein coupled receptors, or GPCRs for short. These receptors are found on the surface of cells and transmit messages across the cell’s outer barrier. When a signaling molecule, like a hormone, is released in the body, it binds to a GPCR and changes the receptor’s shape. The change in structure affects how the GPCR interacts and binds to other proteins on the inside of the cell, triggering a series of reactions that alter the cell’s activity. Scientists have previously seen that a GPCR can trigger different responses depending on which signaling molecule is binding on the surface of the cell. However, the mechanism for this is unknown. One hypothesis is that different signaling molecules change the GPCR’s preference for binding to different proteins on the inside of the cell. The challenge has been to observe this happening without interfering with the process. Stoeber et al. have now tested this idea by attaching fluorescent tags to proteins that bind to activated GPCRs directly and without binding other signaling proteins. This meant these proteins could be tracked under a microscope as they made their way to bind to the GPCRs. Stoeber et al. focused on one particular GPCR, known as the opioid receptor, and tested the binding of two different opioid signaling molecules, etorphine and Dynorphin A. The experiments revealed that the different opioids did affect which of the engineered proteins would preferentially bind to the opioid receptor. This was followed by a similar experiment, where the engineered proteins were replaced with another protein called GRK2, which binds to the opioid receptor under normal conditions in the cell. This showed that GRK2 binds much more strongly to the opioid receptor when Dynorphin A is added compared to adding etorphine. These findings show that GPCRs can not only communicate that a signaling molecule is binding but can respond differently to convey what molecule it is more specifically. This could be important in developing drugs, particularly to specifically trigger the desired response and reduce side effects. Stoeber et al. suggest that an important next step for research is to understand how the GPCRs preferentially bind to different proteins.

KEYWORDS:

GPCR; TIRF; agonist bias; biochemistry; biosensor; cell biology; chemical biology; none; opioid; receptor kinase

Supplemental Content

Full text links

Icon for eLife Sciences Publications, Ltd Icon for PubMed Central
Loading ...
Support Center