Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

The tumor necrosis factor-related apoptosis–inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.     

Identification and characterization of a novel agonistic anti-DR4 human monoclonal antibody

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been established as promising targets for cancer treatment. Therapeutics targeting TRAIL and its receptors are not only effective in killing many types of tumors, but they also synergize with traditional therapies and show efficacy against tumors that are otherwise resistant to conventional treatments. We describe here the identification and characterization of two human monoclonal antibodies, m921 and m922, that are specific for human DR4. Both antibodies competed with TRAIL for binding to DR4, but only m921 recognized cell surface-associated DR4 and inhibited the growth of ST486 cells. This antibody may have potential for further development as a candidate therapeutic and research tool.Key words: DR4, TRAIL, lymphoma, therapeutic antibody     

Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signaling through the DR4 receptor by competing for ligand binding

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a "dominant negative" effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.     

Effect of Diabetes/Hyperglycemia on the Rat Retinal Adenosinergic System

The early stages of diabetic retinopathy (DR) are characterized by alterations similar to neurodegenerative and inflammatory conditions such as increased neural apoptosis, microglial cell activation and amplified production of pro-inflammatory cytokines. Adenosine regulates several physiological functions by stimulating four subtypes of receptors, A₁AR, A_2AAR, A_2BAR, and A₃AR. Although the adenosinergic signaling system is affected by diabetes in several tissues, it is unknown whether diabetic conditions in the retina can also affect it. Adenosine delivers potent suppressive effects on virtually all cells of the immune system, but its potential role in the context of DR has yet to be studied in full. In this study, we used primary mixed cultures of rat retinal cells exposed to high glucose conditions, to mimic hyperglycemia, and a streptozotocin rat model of type 1 diabetes to determine the effect diabetes/hyperglycemia have on the expression and protein levels of adenosine receptors and of the enzymes adenosine deaminase and adenosine kinase. We found elevated mRNA and protein levels of A₁AR and A_2AAR, in retinal cell cultures under high glucose conditions and a transient increase in the levels of the same receptors in diabetic retinas. Adenosine deaminase and adenosine kinase expression and protein levels showed a significant decrease in diabetic retinas 30 days after diabetes induction. An enzymatic assay performed in retinal cell cultures revealed a marked decrease in the activity of adenosine deaminase under high glucose conditions. We also found an increase in extracellular adenosine levels accompanied by a decrease in intracellular levels when retinal cells were subjected to high glucose conditions. In conclusion, this study shows that several components of the retinal adenosinergic system are affected by diabetes and high glucose conditions, and the modulation observed may uncover a possible mechanism for the alleviation of the inflammatory and excitotoxic conditions observed in diabetic retinas.     

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular probes for the human adenosine receptors

Adenosine receptors, G protein–coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.

     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N NMR resonance assignments and secondary structure determination of the extra-cellular domain from the human proapoptotic TRAIL-R2 death receptor 5 (DR5-ECD)

Death receptors (DR) selectively drive cancer cells to apoptosis upon binding to the Tumor necrosis factor-a-Related Apoptosis-Inducing Ligand (TRAIL). Complex formation induces the oligomerization of the death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and transduces the apoptogenic signal to their respective death domains, leading to Death Inducing Signaling Complex (DISC) formation, caspase activation and ultimately cell death. Several crystal structures of the ExtraCellular Domain from Death Receptor 5 (DR5-ECD) have been reported in complex with the TRAIL ligand or anti-DR5 antibodies, but none for the isolated protein. In order to fill this gap and to perform binding experiments with TRAIL peptidomimetics, we have produced isotopically labelled DR5-ECD and started a conformational analysis by using high-field 3D NMR spectroscopy. Herein, we present the first resonance assignment of a TRAIL receptor in solution and the determination of its secondary structure from NMR chemical shifts.     

Nonapoptotic functions of FADD-binding death receptors and their signaling molecules

Death receptors (DRs) are surface receptors that when triggered have the capacity to induce apoptosis in cells by forming the death-inducing signaling complex (DISC). The first protein recruited to form the DISC is the adaptor protein FADD/Mort1. Some members of the DR family, CD95 and the TRAIL receptors DR4 and DR5, directly bind FADD, whereas others, such as TNF receptor I and DR3, initially bind another adaptor protein, TRADD, which then recruits FADD. While all DRs can activate both apoptotic and non-apoptotic pathways, it has been widely assumed that the main physiological role of FADD-binding death receptors is to trigger apoptosis. However, recent work has ascribed multiple non-apoptotic activities to these receptors and/or the signaling components of the DISC.     

Myc Tagging along the TRAIL to Death Receptor 5

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL; also known as Apo2L) is an apoptotic cytokine that is being developed as a novel anticancer agent. TRAIL mediates its effect via death receptors 4 (DR4) and DR5 and appears to selectively induce apoptosis in cancer cells. The molecular basis of why normal cells seem to better tolerate this novel cytokine remains unknown. Recently, it has been reported that Myc oncoprotein by upregulating DR5 appears to augment cellular susceptibility to TRAIL and to DR5 agonistic antibodies. Several previous studies have already established that various clinically relevant agents by upregulating DR5 sensitize cells to TRAIL. However, the finding that DR5 is upregulated by an oncoprotein that is overexpressed in several tumor types is noteworthy and may spark future investigations aiming to explore the Myc and DR5 expression status of primary tumors and their ultimate vulnerability to DR5-targeted therapeutics.     

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.     

Presence of “Ra” and “P”-site receptors for adenosine coupled to adenylate cyclase in cultured vascular smooth muscle cells

The existence of adenosine receptors coupled to adenylate cyclase in cultured vascular smooth muscle cells from rat aorta is demonstrated in these studies. Adenosine, N⁶-phenylisopropyladenosine, adenosine N′-oxide and 2-chloroadenosine stimulated adenylate cyclase in a concentration dependent manner. The stimulation was dependent on the presence of guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In contrast, 2′ deoxyadenosine inhibited adenylate cyclase activity. Adenosine and 2-chloroadenosine showed a biphasic effect on adenylate cyclase, stimulation occurred at low concentrations. The activation of adenylate cyclase by N⁶-phenylisopropyladenosine was also dependent on the Mg²⁺ concentration. The data suggest that vascular smooth muscle cells have both “Ra” and “P” receptors for adenosine, and it can be postulated that the relaxant effect of adenosine on vascular smooth muscle may be mediated by its interaction with “Ra” receptors associated with adenylate cyclase.     

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D) 相似文献   

Comparative binding to DR4 and DR5 receptors of TRAIL and BNNTs/PAHE/mPEG‐DSPE/TRAIL nanoparticles

TRAIL is a member of the tumor necrosis factor family of cytokines, which induces apoptosis of cancer cells, thanks to its binding to its cognate receptors DR5 and DR4. We have recently demonstrated that nanovectorization of TRAIL with single‐walled carbon nanotubes enhanced TRAIL affinity to DR5. In this paper, 1‐pyrenebutyric acid N‐hydroxysuccinimide ester functionalized boron nitride nanotubes (BNNTs) were used to anchor the TRAIL protein. The resulting BNNT/1‐pyrenebutyric acid N‐hydroxysuccinimide ester nanotubes were mixed with methoxy‐poly(ethylene glycol)‐1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐conjugates so as to allow a good dispersion of these nanoparticle TRAIL (NPT) in aqueous solution. The difference of binding between NPT and soluble TRAIL to DR4 and DR5 receptors was then studied by the use of affinity chromatography. DR4 and DR5 receptors were thus immobilized on a chromatographic support, and the binding of the 2 ligands TRAIL and NPT to DR4 and DR5 was studied in the temperature range 30°C to 50°C. Negative enthalpy (ΔH ) values indicated that van der Waals interactions and hydrogen bonding are engaged favorably at the ligand‐receptor interface. It was shown that their rank‐ordered affinities were strongly different in the sequence TRAIL_DR4 < NPT_DR4 < TRAIL_DR5 < NPT_DR5, and the highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on BNNTs. Taken together, our results demonstrated that nanovectorization of TRAIL with BNNTs enhanced its binding to both DR4 and DR5 receptors at 37°C. Our novel nanovector could potentially be used for delivering TRAIL to cells for cancer treatment.     

Adenosine receptors and cancer

Adenosine is a ubiquitous signaling molecule whose physiological functions are mediated by its interaction with four G-protein-coupled receptor subtypes, termed A(1), A(2A), A(2B) and A(3). As a result of increased metabolic rates, this nucleoside is released from a variety of cells throughout the body in concentrations that can have a profound impact on vasculature and immunoescape. However, as high concentrations of adenosine have been reported in cancer tissues, it also appears to be implicated in the growth of tumors. Thus, full characterisation of the role of adenosine in tumor development, by addressing the question of whether adenosine receptors are present in cancer tissues, and, if so, which receptor subtype mediates its effects in cancer growth, is a vital research goal. To this end, this review focuses on the most relevant aspects of adenosine receptor subtype activation in tumors reported so far. Although all adenosine receptors now have an increasing number of recognised biological roles in tumors, it seems that the A(2A) and A(3) subtypes are the most promising as regards drug development. In particular, activation of A(2A) receptors leads to immunosuppressive effects, which decreases anti-tumoral immunity and thereby encourages tumor growth. Due to this behavior, the addition of A(2A) antagonists to cancer immunotherapeutic protocols has been suggested as a way of enhancing tumor immunotherapy. Interestingly, the safety of such compounds has already been demonstrated in trials employing A(2A) antagonists in the treatment of Parkinson's disease. As for A(3) receptors, the effectiveness of their agonists in several animal tumor models has led to the introduction of these molecules into a programme of pre-clinical and clinical trials. Paradoxically, A(3) receptor antagonists also appear to be promising candidates in human cancer treatment of regimes. Clearly, research in this still field is still in its infancy, with several important and challenging issues remaining to be addressed, although purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.     

Identification of a novel DNA binding site for nuclear orphan receptor OR1

The nuclear orphan receptor OR1 has been shown to bind as a heterodimer with retinoid X receptor (RXR) to direct repeat 4 (DR4) response elements. It remained unclear, however, whether this represents the only or the optimal binding site for this receptor. Therefore, we performed a DNA binding site selection assay that allows the identification of novel DNA binding sites for OR1 in an unbiased manner. While OR1 alone was not able to select a specific sequence from the pool of oligonucleotides, the OR1/RXR heterodimer selected a highly conserved DR1 element, termed DR1s, with two AGGTCA motifs spaced by one adenosine. The functional activity of the consensus binding site was verified in transient transfection assays and corroborated by in vitro studies. Based on the sequence of the consensus DR1s, we located putative natural binding sites in the 5'-promoter flanking regions of the rat S14 gene and the rat cholecystokinin type A receptor gene. Furthermore, we could show that although the OR1/RXR heterodimer has a distinct binding orientation on a DR4 element, it is able to bind in both orientations to the DR1s element. The OR1 paralog LXRalpha does not bind as a heterodimer with RXR to the DR1s element, indicating that these receptors, despite their homology, are involved in the regulation of different sets of genes.     

Differential effect of adenosine on tumor and normal cell growth: focus on the A3 adenosine receptor

Adenosine is an ubiquitous nucleoside present in all body cells. It is released from metabolically active or stressed cells and subsequently acts as a regulatory molecule through binding to specific A1, A2A, A2B and A3 cell surface receptors. The synthesis of agonists and antagonists to the adenosine receptors and their cloning enabled the exploration of their physiological functions. As nearly all cells express specific adenosine receptors, adenosine serves as a physiological regulator and acts as a cardioprotector, neuroprotector, chemoprotector, and as an immunomodulator. At the cellular level, activation of the receptors by adenosine initiates signal transduction mechanisms through G-protein associated receptors. Adenosine's unique characteristic is to differentially modulate normal and transformed cell growth, depending upon its extracellular concentration, the expression of adenosine cell surface receptors, and the physiological state of the target cell. Stimulation of cell proliferation following incubation with adenosine has been demonstrated in a variety of normal cells in the range of low micromolar concentrations, including mesangial and thymocyte cells, Swiss mouse 3T3 fibroblasts, and bone marrow cells. Induction of apoptosis in tumor or normal cells was shown at higher adenosine concentrations (>100 microM) such as in leukemia HL-60, lymphoma U-937, A431 epidermoid cells, and GH3 tumor pituitary cell lines. It was further noted that the A3 adenosine receptor (A3AR) plays a key role in the inhibitory and stimulatory growth activities of adenosine. Modulation of the A3AR was found to affect cell growth either positively or negatively depending on the concentration of the agonist, similar to the effect described for adenosine. At nanomolar concentrations, the A3AR agonists possess dual activity, i.e., antiproliferative activity toward tumor cells and stimulatory effect on bone marrow cells. In vivo, these agonists exerted anti-cancer effects, and when given in combination with chemotherapy, they enhanced the chemotherapeutic index and acted as chemoprotective agents. Taken together, activation of the A3AR, by minute concentrations of its natural ligand or synthetic agonists, may serve as a new approach for cancer therapy.     

Adenosine induced apoptosis in BHK cells via P1 receptors and equilibrative nucleoside transporters

Adenosine, as a ubiquitous metabolite, mediates many physiological functions via activation of plasma membrane receptors. Mechanisms of most of its physiological roles have been studied extensively, but research on adenosine-induced apoptosis (AIA) has only started recently. In this study we demonstrate that adenosine dose-dependently triggered apoptosis of cultured baby hamster kidney (BHK) cells. Adenosine-induced apoptotic cell death was characterized by DNA laddering, changes in nuclear chromatin morphology and phosphatidylserine staining. Apoptosis was also quantified by flow cytometry. Results suggest the involvement of adenosine A1 and A3 receptors as well as equilibrative nucleoside transporters in apoptosis induced by adenosine. These results indicate a receptor-transporter co-signaling mechanism in AIA in BHK cells. The involvement of A1 and A3 receptors also implies a possible apoptotic pathway mediated by G protein-coupled receptors.     

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.     

Adenosine receptors: G protein-mediated signalling and the role of accessory proteins.

Ever since the discovery of the effects of adenosine in the circulation, adenosine receptors continue to represent a promising drug target. Firstly, this is due to the fact that the receptors are expressed in a large variety of cells; in particular, the actions of adenosine (or, respectively, of the antagonistic methylxanthines) in the central nervous system, in the circulation, on immune cells and on other tissues can be beneficial in certain disorders. Secondly, there exists a large number of ligands, which have been generated by introducing several modifications in the structure of the lead compounds (adenosine and methylxanthine), some of them highly specific. Four adenosine receptor subtypes have been identified by molecular cloning; they belong to the family of G protein-coupled receptors, which transfer signals by activating heterotrimeric G proteins. It has been appreciated recently that accessory proteins impinge on the receptor/G protein interaction and thus modulate the signalling reaction. These accessory components may be thought as adaptors that redirect the signalling pathway to elicit a cell-specific response. Here, we review the recent literature on adenosine receptors and place a focus on the role of accessory proteins in the organisation of adenosine receptor signalling. These components have been involved in receptor sorting, in the control of signal amplification and in the temporal regulation of receptor activity, while the existence of others is postulated on the basis of atypical cellular reactions elicited by receptor activation.