Micro-vesicles can be released by different cell types and operate as ‘safe containers’ mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release ...exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway.
In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.
The concept of intramembrane receptor-receptor interactions and evidence for their existences were introduced in the beginning of the 1980's, suggesting the existence of receptor heterodimerization. ...The discovery of GPCR heteromers and the receptor mosaic (higher order oligomers, more than two) has been related to the parallel development and application of a variety of resonance energy transfer techniques such as bioluminescence (BRET), fluorescence (FRET) and sequential energy transfer (SRET). The assembly of interacting GPCRs, heterodimers and receptor mosaic leads to changes in the agonist recognition, signaling, and trafficking of participating receptors via allosteric mechanisms, sometimes involving the appearance of cooperativity. The receptor interface in the GPCR heteromers is beginning to be characterized and the key role of electrostatic epitope-epitope interactions for the formation of the receptor heteromers will be discussed. Furthermore, a "guide-and-clasp" manner of receptor-receptor interactions has been proposed where the "adhesive guides" may be the triplet homologies. These interactions probably represent a general molecular mechanism for receptor-receptor interactions. It is proposed that changes in GPCR function (moonlighting) may develop through the intracellular loops and C-terminii of the GPCR heteromers as a result of dynamic allosteric interactions between different types of G proteins and other receptor interacting proteins in these domains of the receptors. The evidence for the existence of receptor heteromers opens up a new field for a better understanding of neurophysiology and neuropathology. Furthermore, novel therapeutic approaches could be possible based on the use of heteromers as targets for drug development based on their unique pharmacology.
The molecular basis for the known intramembrane receptor/receptor interactions among G protein-coupled receptors was postulated to be heteromerization based on receptor subtype-specific interactions ...between different types of receptor homomers. The discovery of GABAB heterodimers started this field rapidly followed by the discovery of heteromerization among isoreceptors of several G protein-coupled receptors such as delta/kappa opioid receptors. Heteromerization was also discovered among distinct types of G protein-coupled receptors with the initial demonstration of somatostatin SSTR5/dopamine D2 and adenosine A1/dopamine D1 heteromeric receptor complexes. The functional meaning of these heteromeric complexes is to achieve direct or indirect (via adapter proteins) intramembrane receptor/receptor interactions in the complex. G protein-coupled receptors also form heteromeric complexes involving direct interactions with ion channel receptors, the best example being the GABAA/dopamine D5 receptor heteromerization, as well as with receptor tyrosine kinases and with receptor activity modulating proteins. As an example, adenosine, dopamine, and glutamate metabotropic receptor/receptor interactions in the striatopallidal GABA neurons are discussed as well as their relevance for Parkinson's disease, schizophrenia, and drug dependence. The heterodimer is only one type of heteromeric complex, and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist. These complexes may assist in the process of linking G protein-coupled receptors and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for some forms of learning and memory.
The quite recent (at least on the evolutionary time scale) emergence of nervous systems in complex organisms enabled the living beings to build a wide-ranging model of the external world in order to ...predict and evaluate the outcomes of their actions. Such a process likely represents a real coding activity, since, by proper handling of information, it generates a mapping between the external environment and internal cerebral activity patterns. The patterns of neural activity that correspond to the final maps, however, emerge from the holistic assembly of a multilevel functional organization. Nerve tissue components, indeed, appear organized in compartments, also called functional modules (FM), that contain system components and circuits of different miniaturizations not only arranged to work together either in parallel or in series but also nested within each other. At least three levels can be recognized in a functional module and it is possible to point out that such a hierarchical organization of the brain circuits could be mirrored by a corresponding hierarchical organization of biocodes. This feature can also suggest the hypothesis that the same logic could operate also at system level to integrate FM into functional brain areas and to associate areas to generate the final map used by humans to image the external world and to imagine untestable worlds.
Several types of D2R and D1R heteroreceptor complexes were discovered in the indirect and direct pathways of the striatum, respectively. The hypothesis is given that changes in the function of the ...dopamine heteroreceptor complexes may help us understand the molecular mechanisms underlying the motor complications of long-term therapy in Parkinson's disease (PD) with l-DOPA and dopamine receptor agonists.
In the indirect pathway, the potential role of the A2AR-D2R, A2AR-D2R-mGluR5 and D2R-NMDAR heteroreceptor complexes in PD are covered and in the direct pathway, the D1R-D3R, A1R-D1R, D1R-NMDAR and putative A1R-D1R-D3R heteroreceptor complexes.
One explanation for the more powerful ability of l-DOPA treatment versus treatment with the partial dopamine receptor agonist/antagonist activity to induce dyskinesias, may be that dopamine formed from l-DOPA acts as a full agonist. The field of D1R and D2R heteroreceptor complexes in the CNS opens up a new understanding of the wearing off of the antiparkinson actions of l-DOPA and dopamine receptor agonists and the production of l-DOPA-induced dyskinesias. It can involve a reorganization of the D1R and D2R heteroreceptor complexes and a disbalance of the D1R and D2R homomers versus non-dopamine receptor homomers in the direct and indirect pathways.
Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be ...transferred by means of MVs and TNTs from a source cell to target cells.
Western blot, transmission electron microscopy and gene expression analyses demonstrate that A2A and D2 receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D2R-CFP or A2AR-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D2R-CFP and A2AR-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs.
Finally, recipient cells pre-incubated for 24h with A2AR positive MVs were treated with the adenosine A2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A2ARs were functionally competent in target cells.
These findings demonstrate that A2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.
Adenosine–dopamine interactions in the central nervous system (CNS) have been studied for many years in view of their relevance for disorders of the CNS and their treatments. The discovery of ...adenosine and dopamine receptor containing receptor mosaics (RM, higher‐order receptor heteromers) in the striatum opened up a new understanding of these interactions. Initial findings indicated the existence of A2AR‐D2R heterodimers and A1R‐D1R heterodimers in the striatum that were followed by indications for the existence of striatal A2AR‐D3R and A2AR‐D4R heterodimers. Of particular interest was the demonstration that antagonistic allosteric A2A‐D2 and A1‐D1 receptor–receptor interactions take place in striatal A2AR‐D2R and A1R‐D1R heteromers. As a consequence, additional characterization of these heterodimers led to new aspects on the pathophysiology of Parkinson's disease (PD), schizophrenia, drug addiction, and l‐DOPA‐induced dyskinesias relevant for their treatments. In fact, A2AR antagonists were introduced in the symptomatic treatment of PD in view of the discovery of the antagonistic A2AR–D2R interaction in the dorsal striatum that leads to reduced D2R recognition and Gi/o coupling in striato‐pallidal GABAergic neurons. In recent years, indications have been obtained that A2AR‐D2R and A1R‐D1R heteromers do not exist as heterodimers, rather as RM. In fact, A2A‐CB1‐D2 RM and A2A‐D2‐mGlu5 RM have been discovered using a sequential BRET‐FRET technique and by using the BRET technique in combination with bimolecular fluorescence complementation. Thus, other pathogenic mechanisms beside the well‐known alterations in the release and/or decoding of dopamine in the basal ganglia and limbic system are involved in PD, schizophrenia and drug addiction. In fact, alterations in the stoichiometry and/or topology of A2A‐CB1‐D2 and A2A‐D2‐mGlu5 RM may play a role. Thus, the integrative receptor–receptor interactions in these RM give novel aspects on the pathophysiology and treatment strategies, based on combined treatments, for PD, schizophrenia, and drug addiction.
Recently evidence has been presented that adenosine A2A and dopamine D2 receptors form functional heteromeric receptor complexes as demonstrated in human neuroblastoma cells and mouse fibroblast Ltk- ...cells. These A2A/D2 heteromeric receptor complexes undergo coaggregation, cointernalization, and codesensitization on D2 or A2A receptor agonist treatments and especially after combined agonist treatment. It is hypothesized that the A2A/D2 receptor heteromer represents the molecular basis for the antagonistic A2A/D2 receptor interactions demonstrated at the biochemical and behavioral levels. Functional heteromeric complexes between A2A and metabotropic glutamate 5 receptors (mGluR5) have also recently been demonstrated in HEK-293 cells and rat striatal membrane preparations. The A2A/mGluR5 receptor heteromer may account for the synergism found after combined agonist treatments demonstrated in different in vitro and in vivo models. D2, A2A, and mGluR5 receptors are found together in the dendritic spines of the striatopallidal GABA neurons. Therefore, possible D2/A2A/mGluR5 multimeric receptor complexes and the receptor interactions within them may have a major role in controlling the dorsal and ventral striatopallidal GABA neurons involved in Parkinson's disease and in schizophrenia and drug addiction, respectively.
G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in ...a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.
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