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 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 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. Consequently, the GPCR-mediated 14-3-3 signaling pathway provides the potential for understanding GPCR signaling pathways more deeply, as well as for the development of novel strategies that target specific GPCR signaling pathways.

GPCR_14-3-3 LinkLight Assay

Targeting specific signaling pathways is a new frontier for GPCR drug development.  Evidence21 from publications suggested that opioid-induced analgesia of the μ-opioid receptor (MOR) results from the G protein signaling, while side effects, including respiratory depression and constipation, may work through the β-arrestin signaling pathway22. Biased ligands like PZM21 that selectively trigger G-protein signaling over β -arrestin signaling open up the possibility of developing potent analgesics without harmful respiratory effects.  Indeed, in a mouse study, PZM21 showed analgesic effects without adverse effects classically observed with morphine. However, it would be overly simplistic to attribute all the side effects to the β-arrestin signaling pathway; β-arrestin knockout mice seem to have a stronger preference for morphine over saline23,24, suggesting drug addiction may not be tied to the β-arrestin signaling pathway. Very likely, there are other MOR signaling pathways we still do not know about. Thus, investigating other potential GPCR signaling mechanisms and pharmacological profiles of GPCR ligands on various signaling pathways including the 14-3-3 pathway could present new opportunities for more targeted drug development.


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