A Generic Approach for the Purification of Signaling Complexes That Specifically Interact with the Carboxyl-terminal Domain of G Protein-coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His 6 -tagged baits, immobilized metal affinity chromatography, one-and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT 1 and MT 2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT 1 , 14 proteins that interacted with the C-tail of MT 2 , and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT 1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques. Molecular & Cellular Proteomics 7: 1556 -1569, 2008.

Melatonin receptor structures shed new light on melatonin research

“…Consistent with the high degree of sequence homology, the overall structures and the binding pose of nonselective melatonin receptor agonists proved to be very similar for both receptors (Figure A,B). Interestingly, this high structural conservation is also observed for helix VIII, which has been identified as a hotspot for interaction with signaling molecules, in particular for MT 1 . It is important to note that all crystal structures of both receptors represent the inactive, low‐affinity receptor state, despite the presence of melatonin receptor agonists.…”

Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

“…All of these GPCRs were studied in brain tissue where GPCRs and their binding partners are highly expressed, enabling isolation of sufficient quantities of the receptor and associated proteins. Furthermore, studies with 5-HT receptors and α 2 B-AR used c-terminal peptides of the receptors (not the full-length GPCR) as baits [21], [23], [24], [26], [27]. Therefore, those studies cannot identify binding partners that interact with GPCR domains outside of the c-terminus.…”

Melatonin and Pancreatic Islets: Interrelationships between Melatonin, Insulin and Glucagon

“…According to these data a picture emerges in which signaling via melatonin receptors involves a plethora of scaffold proteins in addition to G-proteins. Maurice et al [82] identified no less than 22 proteins capable of interacting with the carboxy-terminal tail of MT1, and 14 proteins associated with MT2. Supporting these results, the interaction between one of the regulators of G-protein signaling (RGS20), which represents another group of multifunctional signaling proteins, and MT1 has been proven [83].…”