**Application of Quantitative Immunogold Electron Microscopy to Determine the Distribution and Relative Expression of Homo- and Heteromeric Purinergic Adenosine A1 and P2Y Receptors**

Kazunori Namba

*National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo Japan* 

### **1. Introduction**

188 Biochemistry

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31249-31256.

The idea that G-protein-coupled receptors (GPCRs) may generate or modify various functions as dimmers or higher-order oligomers is now generally accepted. Significant numbers of GPCRs exit as heteromeric assemblies (refered to as hetero-oligomerization), generating novel functions for ligand binding and second messengers, and in turn creating unique receptor trafficking systems for pharmacological profiles (Angers *et al*., 2002, Bulenger *et al*., 2005). This is also true of the purinergic receptor family. Over recent years, we have explored many biochemical and pharmacological aspects of this particular family via hetero-oligomerization between metabotropic (i.e. G protein-coupled) purinergic receptors (particularly between P1 and P2), in which the agonists are metabolites playing important role in the purinergic signaling cascade.

Purines such as adenosine triphosphate (ATP), via their specific P1 and P2 receptors, mediate a variety of physiological processes including pathophysiology, neurotransmission, neuromodulation, pain, cardiac function, immune responses and almost every aspect of development (Abbracchio *et al*., 2009; Burnstock, 2007; Burnstock, 2008; Ralevic *et al*., 1998). P1 receptors are further sub-classified into A1, A2A, A2B and A3 sub-types, all of which are G protein-coupled receptors (GPCRs). The adenosine A1 receptor (A1R) is known to regulate Ca2+/K+ channels, adenylate cyclase, and phospholipase C by coupling to Gi/o proteins (Ralevic *et al*., 1998). The P2 receptors can be further sub-classified into ligand-gated ion channel-type P2X(1-7) receptors, and G protein-coupled P2Y(1, 2, 4, 6, 11, 12, 13, 14) receptors. P2Y2Rspecific pharmacology (induction of Ca2+ release) has been analyzed in detail using CHO-K1 cells (Mehta *et al*., 2008). In hippocampal astrocytes, P2Y1R- and P2Y2R-mediated Ca2+ responses differentially show two forms of activity-dependent negative feedback of synaptic transmission via the phospholipase C beta-IP3 pathway (Fam *et al*., 2003). P2Y2 R modulation of pain responses has also been reported (Molliver *et al*., 2002). Today, homo- or heterooligomers of many kinds of GPCRs have been reported (Bouvier, 2001) and the heterooligomerization of GPCRs affects various aspects of receptor function, including the alteration of ligand-binding specificity and cellular trafficking. We previously demonstrated that A1R associates with P2Y1R in co-transfected HEK293T cells and in rat brain homogenates, whereby a P2Y1R agonist stimulates A1R signaling via Gi/o (Yoshioka *et al*., 2001, Yoshioka *et al*., 2002). Furthermore, in co-transfected HEK293T cells, hetero-oligomers display unique pharmacology whereby simultaneous activation of the two receptors attenuates A1R signaling via Gi/o, but synergistically enhances P2Y2R signaling via Gq/11 (Suzuki *et al*., 2006). Because A1R are widely expressed in the brain (Yoshioka *et al*., 2002), it is likely that these receptors also associate directly *in situ*; however, direct evidence of their oligomerization or precise co-localization in brain has yet to be demonstrated. In our laboratory, we are developing a new method, immunogold electron microscopic observation using different sized-immunogold particles enable visualize the oligomerization of A1R and P2Y2R (Namba *et al*., 2010). The aim of the study was to determine whether A1R and P2Y2R associate with each other in the rat brain by looking for receptor complexes with immunogold electron microscopy (IEM). This method also provides information concerning the localization and density of GPCR monomers and oligomers expressed in transfected cells, that are also applicable to tissues such as brain.

In this chapter, we describe both pre- and post-embedding electronmicroscopic techniques to identify cells or tissues expressing GPCRs utilizing differently-sized immunogold particles, and review IEM quantification as an efficient approach to analyze two specific types of data. One data set represents the classification of receptor formations. A1R and P2Y1R (P2Y2R) produce five receptor formations which are made up of monomers (A1R, P2YR), homo-oligomers (A1R- A1R, P2YR- P2YR) and hetero-oligomers (A1R-P2YR). The second dataset describes the estimation of receptor expression levels by counting immunoreactive immunogold particles at the cell surface.

Establishing specific expression patterns of GPCRs at the ultrastructural level, and detecting homo- and hetero-oligomers of GPCRs in both co-transfected cultured cells and tissues, will enable us to visually understand some of the phenomena underlying signal transduction signalling pathways operating via GPCRs in a heteromeric dependent manner. It is widely accepted that drug discovery targets for rapid remedies are likely to be specific receptors expressed upon the cytoplasmic membrane. In order to establish the precise effects of new drugs, the expression patterns and expression level of A1R and P2Y1R (P2Y2R) represent significant factors to be considered, especially with regard to their association with cross-talk systems.
