C The membrane permeable crosslinker DSP, on the other hand, can be used to crosslink receptors and their interacting proteins in living cells.
A number of crosslinkers have been used to study receptor—protein interactions in cells Brenner et al. These compounds are characterized by differences in their spacer arm as well as the composition of the two amine binding groups that recognize and covalently bind specific functional groups on target proteins Figures 1B,C ; Sinz, ; Trakselis et al. Table 1 lists crosslinkers that have been used to study receptor binding to intracellular proteins.
A recent study utilized agarose beads whose surface was covalently linked with a cleavable chemical crosslinker by spacers of varied lengths to study the interactome of the post-synaptic density isolated from the rodent cortex Yun-Hong et al. Experiments successfully demonstrate assemblages of proteins at various subcellular distances and compartments, including the post-synaptic density, thus underscoring the utility of the approach in the characterization of interactomes based on spacer arm properties.
However, a possible disadvantage of CC is that some antibodies are no longer able to recognize their target protein after crosslinking Boudreau et al. A summary of crosslinkers used in the analysis of receptor protein interactions. Interaction with trafficking and chaperone proteins is important for directing the localization and function of the receptor Jeanclos et al.
Protein associations may also contribute to receptor conformation at the cell surface Giniatullin et al. While the ability to detect receptors at the plasma membrane and in the cytosol has been traditionally reliant on epitope tagging, live cell stain, and cell surface labeling methods such as biotinylation, CC has emerged as a complementary tool in the study of interacting proteins responsible for receptor cellular trafficking and localization.
The study interestingly demonstrates that receptor signaling is sustained even in the absence of ligand binding. Crosslinking has also enabled detection of changes in receptor function for the angiotensin receptor Quian et al. When combined with cell surface labeling, CC has been effective in determining changes in receptor glycosylation.
In this study CC revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMP — suggestive of a direct association between the receptor and the accessory protein during ligand activation. Nicotinic acetylcholine receptors nAChRs are a family of ligand gated ion channels expressed throughout the nervous system contributing to learning, memory, and goal driven behavior Changeux, Recent evidence also reveals that nAChRs operate by coupling to intracellular proteins such as heterotrimeric G proteins Kabbani et al.
Chronic nicotine exposure gives rise to neural adaptations such as an up-regulation of specific nAChRs through cell-delimited post-translational mechanisms Sallette et al. These receptor mechanisms are a hallmark of nicotine addiction yet it is still unclear which signaling pathways and mechanism regulate nAChR assembly and trafficking inside the cell.
Proteomic studies, based on yeast-two-hybrid as well as conventional IP experiments have led to the identification of several intracellular proteins that bind nAChR subunits in the brain Kabbani et al. Directed protein interaction screens have also enabled discovery of proteins responsible for nAChR trafficking and assembly Lin et al. In these studies CC was vital to the detection of changes in receptor interaction with signaling molecules and heterotrimeric G proteins.
The CC method was also able to enhance the detection of small signaling molecules such as receptor kinases in both Western blots and mass spectrometry experiments Hu et al. Identifying protein interactions that mediate nAChR trafficking, localization, and signaling. Alternatively, antibody labeling and crosslinking can be used to examine changes in the receptor interactome between internalized and cell surface nAChRs.
B Chemical crosslinking can be used to study the dynamics of nAChR—protein interactions under various ligand treatment conditions. Proteomic and yeast-two-hybrid studies on receptor interactions have enabled a broad understanding on the diversity and function of receptor—protein interactions in cells. These studies have enabled an interaction-based framework for defining the mechanisms of receptor signaling.
Receptor—protein interaction identification however is not sufficient for understanding how receptors operate in cells. In particular, important questions remain on the spatial specificity and temporal aspects of receptor expression and signaling in cells. For multi-subunit channel receptors such as the glutamate AMPA receptor, the addition of the membrane impermeable linker BS 3 has proven effective in the analysis of receptor subunit composition at the cell surface Boudreau et al.
Similar approaches with the aim of detecting protein—protein interaction in living cells are now necessary. Advancement in the design and experimental utility of CC such as photo-reactive amino acid analogs Suchanek et al. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Src-mediated tyrosine phosphorylation of dynamin is required for beta 2-adrenergic receptor internalization and mitogen-activated protein kinase signaling.
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Pubmed Abstract Pubmed Full Text. Desensitization of nicotinic ACh receptors: Affinity labelling of steroid hormone receptors. An integrated mass spectrometry-based proteomic approach: Structural basis of G protein-coupled receptor-G protein interactions.
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RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Transduction of receptor signals by beta-arrestins. The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor. Protozoa and Human Disease Mark F. Power, Sex, Suicide Nick Lane. A Dictionary of Biology Robert C.
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