抄録
Background
GPCRs are responsible for many important physiological processes, are the target of over 30% of current medicines, and their continuous stimulation can lead to various disease states: b2-adrenoceptor (b2AR) has been implicated in cancer metastasis, whereas prolonged activation of the mu-opioid receptor (MOPr) is linked to addiction and tolerance. We used imaging approaches with high spatio-temporal resolution and quantitative mass spectrometry to show that signalling of these receptors is driven by the assembly of distinct multi-protein complexes.
Methods
To investigate the formation and composition of b2AR- or MOPr-mediated complexes we used Forster Resonance Energy Transfer (FRET) and immunoprecipitation. FRET-based cAMP, PKC and ERK biosensors were transiently expressed in HEK293 cells and used to measure the responses of endogenous b2AR to sub-picomolar concentrations of isoprenaline and of MOPr to DAMGO ([D-Ala2,N-MePhe4, Gly5-ol]-enkephalin) and morphine. Internalisation and protein translocation were measured by Bioluminescence Resonance Energy Transfer (BRET) and Total Internal Relflection Fluorescence (TIRF) microscopy, respectively. Quantitative mass-spectrometry was used to measure proteomic changes upon ligand stimulation.
Results
b2AR assembles into a large protein complex, allowing responses to femtomolar concentrations of agonist which are different to responses to normal (nanomolar) concentrations and are dependent on constitutive receptor recycling. Quantitative mass spectrometry revealed that this difference results in induction of distinct proteomic profiles; only femtomolar isoprenaline causes an increase in global gene transcriptional activity. The MOPr displays ligand-dependent spatio-temporal signalling. Morphine activates plasma membrane-localised PKC, which prevents receptor translocation within the plasma membrane, resulting in sustained cytosolic ERK activity and no nuclear ERK activity. Activation of MOPr with DAMGO triggers fast receptor translocation within the plasma membrane, precedes trafficking to clathrin-containing domains and internalisation, and is likely dependent on receptor phosphorylation. This results in transient cytosolic and nuclear ERK activation.
Conclusions
Our results suggest that GPCRs form highly specific protein complexes that mediate unique responses. We are exploring this further by using b2AR with C-terminal biotin ligase (BirA) and a MOPr with a C-terminal ascorbate peroxidase (APEX2) to identify new interacting proteins that participate in these specific signalling profiles, not only in recombinant cell lines but also in more physiological systems.
