GPCR/14-3-3 Signaling Pathway Assays

GPCR signal transduction is pluridimensional. In addition to well-documented G protein-dependent and β-arrestin-dependent GPCR signaling pathways, other cellular effectors are recruited to GPCRs. These proteins form signal complexes with GPCRs to elicit specific signaling cascades.  Multiple cellular effectors offer the possibility of crosstalk, fine-tuning and specifically regulating GPCR signaling at multiple cascades1,2.

One of the recruited proteins is a multifunctional signal adaptor protein 14-3-3. 14-3-3 proteins are ~30kDa proteins, ubiquitously expressed in eukaryotic cells, and their highest expression is found in the brain, where they make up approximately 1% of total soluble protein. In neurons, 14-3-3 proteins are present in the cytoplasm, intracellular organelles, and plasma membrane. Some of the 14-3-3 isoforms are particularly enriched in synapses, presumably to regulate neurotransmission and plasticity3.   Although lacking enzymatic activity, 14-3-3 proteins bring two or more proteins together to transduce signals. What is so remarkable about 14-3-3 proteins is the number and diversity of proteins they interact with4.  Interaction partners include kinases, phosphatases, ubiquitin ligases, transcription factors, scaffold proteins, cytoskeletal proteins, and membrane proteins including GPCRs, receptor tyrosine kinases (RTKs), cytokine receptors, and ion channels5-16.  The interactions facilitate the formation of large molecular complexes that coordinate responses of multiple signaling pathways to incoming stimuli, allowing signal transduction among different cellular compartments, and carrying out a variety of physiological functions.  Not surprisingly, 14-3-3 proteins are also associated with human diseases, most notably in cancers and neurological disorders such as Alzheimer’s disease, Parkinson’s disease, spinocerebellar ataxia type 1, schizophrenia, and bipolar disorder based on evidence from both clinical and laboratory studies17-20.

Although biochemical evidence shows 14-3-3 forms complexes with some GPCRs5-11, investigation of GPCR-mediated 14-3-3 signaling has been largely ignored.  Lack of ability to assess specific 14-3-3 signaling is a major reason.  Given the facts that 14-3-3 proteins are highly expressed in the brain and involved in numerous cellular processes, development of tools for pharmacologically characterizing GPCR-mediated 14-3-3 signaling is a must.  Such tools not only aid us in understanding GPCR signaling pathways but also allow us to for screen molecules modulating the 14-3-3 signaling pathway.

We have successfully demonstrated that 14-3-3 signal adaptor proteins, like G-proteins and β-arrestins, interact with GPCRs in a ligand-dependent manner, and for the first time, we can pharmacologically characterize GPCR-mediated 14-3-3 signaling pathways. Development of a direct measure of GPCR-mediated 14-3-3 signaling and that we can pharmacologically characterize ligands modulating this pathway opens up a new realm of previously unappreciated GPCR signal transduction.

GPCR_14-3-3 LinkLight Assay

Receptor trafficking and recycling are crucial for the temporal and spatial control of GPCR signaling. We found that GPCRs interact with 14-3-3 signal adaptor/scaffold proteins and that this interaction regulates receptor trafficking and recycling. GPCR-mediated 14-3-3 signaling pathway provides the potential for the development of novel strategies that target specific GPCR signaling pathways.

BioInvenu now offers “target-open” assay systems for researchers to develop their-own interested target LinkLight assays. The system contains multiple 14-3-3 isoform reporter host cell lines and “target-open” cloning vectors. Please contact info@bioinvenu.com for detail information. 14-3-3 proteins are known to interact with a huge number of different proteins.  Interaction partners include kinases, phosphatases, scaffold proteins, transcription factors, cytoskeletal proteins, ubiquitinases, and membrane proteins including GPCRs, RTKs, and ion channels.

https://www.nature.com/articles/s41598-019-47478-w

https://www.nature.com/articles/sigtrans201618

http://bioinvenu.com/wp-content/uploads/2017/05/Poster_GPCR_14-3-3.pdf

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