An activity-based probe reveals dynamic protein–protein interactions mediating IGF-1R transactivation by the GABAB receptor

Abstract

Many GPCRs (G-protein-coupled receptors) can activate RTKs (receptor tyrosine kinases) in the absence of RTK ligands, a phenomenon called transactivation. However, the underlying molecular mechanisms remain undefined. In the present study we investigate the molecular basis of GABA(B) (γ-aminobutyric acid B) receptor-mediated transactivation of IGF-1R (insulin-like growth factor type I receptor) in primary neurons. We take a chemical biology approach by developing an activity-based probe targeting the GABA(B) receptor. This probe enables us first to lock the GABA(B) receptor in an inactive state and then activate it with a positive allosteric modulator, thereby permitting monitoring of the dynamic of the protein complex associated with IGF-1R transactivation. We find that activation of the GABA(B) receptor induces a dynamic assembly and disassembly of a protein complex, including both receptors and their downstream effectors. FAK (focal adhesion kinase), a non-RTK, plays a key role in co-ordinating this dynamic process. Importantly, this dynamic of the GABA(B) receptor-associated complex is critical for transactivation and transactivation-dependent neuronal survival. The present study has identified an important mechanism underlying GPCR transactivation of RTKs, which was enabled by a new chemical biology tool generally applicable for dissecting GPCR signalling.

Smoking-associated lung cancer prevention by blockade of the beta-adrenergic receptor-mediated insulin-like growth factor receptor activation

“…It has been proposed that various RTKs, including EGFR and IGF-1R, can be transactivated by GPCRs via several mechanisms, including ligand-dependent activation by metalloproteinase-dependent shedding of ligands and reactive oxygen species (ROS)- or Src-mediated ligand-independent activation [26]. RTK transactivation via direct association between RTKs and GPCRs through the formation of a complex with other non-receptor tyrosine kinases, adaptor proteins, and β-arrestin has also been proposed [27, 28]. Our results demonstrate that NNK treatment leads to increased IGF2 transcription via activating β-AR, STAT3, and NF-κB.…”

GABAB receptor promotes its own surface expression by recruiting a Rap1-dependent signaling cascade

“…As no proteins known to bind to the C-terminus of the GB1 or GB2 subunits appear to regulate the cell surface stability of GABA B receptor (Benzing et al, 2000;Brock et al, 2005;Guetg et al, 2010;Kuramoto et al, 2007;Nehring et al, 2000;Schwenk et al, 2010;Tan et al, 2004), we set out to identify new GABA B -receptor-interacting proteins. To this end, we chose to work with cerebellar granule neurons (CGNs) that express endogenous GABA B receptor, as the function of GABA B receptor has been well characterized by us and many others in these neurons (Lin et al, 2012;Tu et al, 2007Tu et al, , 2010. We stimulated CGNs with the prototypical GABA B receptor agonist baclofen, and then used glutathione-S-transferase (GST) fusion proteins containing the C-terminal part of GB1 or GB2 (GST-GB1-C or GST-GB2-C) Fairfax et al, 2004;Kuramoto et al, 2007;Nehring et al, 2000;Perroy et al, 2003) as a bait to pull down potential interacting proteins from the cell lysate (Fig.…”

Regulation of autophagy and mitophagy by nutrient availability and acetylation

“…Interestingly, one family of deacetylases linked to caloric-restriction mediated longevity, the sirtuins or Sirts, employ NAD as a cofactor to mediate lysine residue deacetylation. The nutrient-sensing enzymes that regulate protein acetylation/deacetylation have begun to be characterized [15, 16], and of note, the mitochondrial enriched deacetylase Sirt3 is itself regulated in response to changing nutrient levels [17-19]. Despite the accumulating evidence supporting the role of this pathway in the modulation of mitochondrial function, this biology requires additional study as epitomized by the lack of metabolic phenotype in specific genetic depletion of Sirt3 in tissues such as the liver and skeletal muscle [20].…”