**9. References**

200 Biochemistry

ADP is a strong agonist for P2Y1R but not P2Y2R (Abbracchio *et al*., 2006). Many previous studies suggest that A1R-P2Y1R and A1R-P2Y2R hetero-oligomers exhibit general pharmacological profiles, possibly because of differences in the conformational changes induced by oligomerization (Nakata *et al*., 2010). The hetero-oligomerization of A1R-P2Y1R inhibits adenylyl cyclase activity via the Gi/o protein linked effector. The heterooligomerization of A1R-P2Y2R resulted in an increase in intracellular Ca2+ levels induced by P2Y2R activation of Gq/11 which was synergistically enhanced by the simultaneous addition of an A1R agonist in the co-expressing cells (Suzuki *et al*., 2006). Differences in the amounts of hetero-oligomerization between A1R-P2Y1R and A1R-P2Y2R were observed (Fig. 7). Assuming that the number of hetero-oligomers formed is functionally dominant, the dominancy of the signaling via A1R-P2Y1R may be generated by competitive antagonism in pharmacology between P2Y1R and P2Y2R in order to oligomerize with A1R. This hypothesis,

In our previous study, the hippocampal hetero-oligomerization of A1R and P2Y2R was far more pronounced than in other regions of the brain (Namba *et al*., 2010). Another research group suggested that the hetero-oligomerization, or cross-talk between A1R and P2Y1R is involved in regulation of glutamate release in the hippocampus (Tonazzini *et al*., 2007). The relative distributions of immunoreactivity for GABAB R2 and GABAB R1 were also different in the basal ganglia and globus pallidus/substantia nigra, which suggests the possible coexistence and hetero-oligomerization of the two types of receptors at various pre- /postsynaptic sites (Charara *et al*., 2004). From the present study, it can be speculated that the A1R/P2Y2R hetero-oligomer might be responsible for down regulation, via hippocampal Ca2+ secretion, of synaptic functions (Safiulina *et al*., 2006). Furthermore, the abundant formation of A1R/P2Y1R or A1R/P2Y2R hetero-oligomers in the cerebellum revealed in this present study supports the idea that the unique signal transduction generated by heterooligomerization, including the enhancement of Ca2+ signaling via Gq/11, observed in

There are many families of GPCRs expressed in whole brain, most of which remain a mystery. However, it is clear that GPCR hetero-oligomerization is common in the brain and exhibits unique pharmacology in this region, thus implying that associated signal transduction pathways can be anticipated in this region. The methodology described here using immunogolod particles is one of the most influential techniques available to elucidate

In summary, IEM provided direct evidence for the existence of homo- and hetero-oligomers of A1R and P2Y2R, not only in co-transfected cultured cells, but also *in situ* on the surface of neurons in various brain regions. The molecular mechanisms responsible for the control of A1R and P2YR monomer/homo-oligomer/hetero-oligomer ratios remain to be elucidated. Future investigation of GPCR oligomer formation is indispensable for revealing the

The importance of these novel experimental procedures using IEM is to provide information concerning crosstalk between small molecules with high angle views of whole cells, although these methods do require a high level of technical skill. The development of ingenious histochemical and immunoelectronmicroscopic methods has made it possible to

however, requires further investigation.

transfected cells, also occurs in the cerebellum.

elaborate mechanisms of cellular function.

**7. Summary** 

the ingenious mechanism underlying GPCR hetero-oligomerization.


**Part 2** 

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202 Biochemistry

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