In Silico Analyses of Vertebrate G-Protein-Coupled Receptor Fusions United With or Without an Additional Transmembrane Sequence Indicate Classification into Three Groups of Linkers.

Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)-(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI-L-SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates.

Synthesis, characterization, low temperature solid state PL and photocatalytic activities of Ag2O·CeO2·ZnO nanocomposite.

A novel multi-metal nanocomposite oxide Ag2O·CeO2·ZnO has been prepared by co-precipitation of their carbonates from aqueous solutions of the metal nitrates following calcinations and annealing 5h at 450°C and 4h at 600°C. Ag2O·CeO2·ZnO has been characterized by XRD, SEM, EDS and PL spectra. According to XRD results the crystallite size of Ag2O·CeO2·ZnO varies in the range of 19-111 nm with an average size of 50 nm, which is in good agreement with SEM results. Elemental analysis was performed by SEM-EDS. Emissions of Ag2O·CeO2·ZnO has been observed in UV (NBE emission), visible and NIR regions at 325 nm excitation by a line of He-Cd laser. Photocatalytic as well as anti-bacterial activities have been studied. The nano composite Ag2O·CeO2·ZnO shows an excellent photocatalytic dye degradation activity.

Analysis of various types of single-polypeptide-chain (sc) heterodimeric A2AR/D2R complexes and their allosteric receptor-receptor interactions.

Adenosine A2A receptor (A2AR) heteromerizes with dopamine D2 receptor (D2R). However, these class A G protein-coupled receptor (GPCR) dimers are not fully formed, but depend on the equilibrium between monomer and dimer. In order to stimulate the heteromerization, we have previously shown a successful design for a fusion receptor, single-polypeptide-chain (sc) heterodimeric A2AR/D2R complex. Here, using whole cell binding assay, six more different scA2AR/D2R constructs were examined. Not only in scA2AR/D2R 'liberated' with longer spacers between the two receptors, which confer the same configuration as the prototype, the A2AR-odr4TM-D2LR, but differ in size (Forms 1-3), but also in scA2AR/D2LR (Form 6) fused with a transmembrane (TM) of another type II TM protein, instead of odr4TM, neither of their fixed stoichiometry (the apparent ratios of A2AR to D2R binding sites) was 1, suggesting their compact folding. This suggests that type II TM, either odr4 or another, facilitates the equilibrial process of the dimer formation between A2AR and D2LR, resulting in the higher-order oligomer formation from monomer of scA2AR/D2LR itself. Also, in the reverse type scA2AR/D2LR, i.e., the D2LR-odr4TM-A2AR, counter agonist-independent binding cooperativity (cooperative folding) was found to occur (Forms 4 and 5). In this way, the scA2AR/D2LR system has unveiled the cellular phenomenon as a snapshot of the molecular behavior in A2AR/D2LR dimer. Thus, these results indicate that the various designed types of functional A2AR/D2R exist even in living cells and that this fusion expression system would be useful to analyze as a model of the interaction between class A GPCRs.

NIR and CT luminescence spectra of [Yb(TFN)(S-BINAPO)] and [Yb(HFA)(S-BINAPO)] complexes.

The complexes [Yb(TFN)3(S-BINAPO)](TFN=4,4,4-trifluoro-1(2-napthyl)-1,3-butanedione) (complex 1) and [Yb(HFA)3(S-BINAPO)](HFA=hexafluoroacetylacetonate) (complex 2) were synthesized, characterized. The absorption as well as PL spectra have been studied. The complex [Yb(TFN)3(S-BINAPO)] showed narrowed emission peak (half width ∼6 nm) at around 981 nm in addition to several emission peaks in NIR (near infrared) region. The complex [Yb(HFA)3(S-BINAPO)] showed strong emission peak at around 985 nm. The charge transfer luminescence of [Yb(TFN)3(S-BINAPO)] was also observed at 412-463 nm.

Hetero-oligomerization and specificity changes of G protein-coupled purinergic receptors: novel insight into diversification of signal transduction.

The formation of homo- and hetero-oligomers between various G protein-coupled receptors (GPCRs) has been demonstrated over the past decade. In most cases, GPCR heterodimerization increases the diversity of intracellular signaling. GPCR-type purinergic receptors (adenosine and P2Y receptors) are actively reported to form hetero-oligomers with each other, with GPCRs belonging to the same group (type 1, rhodopsin-like), and even with GPCRs from another group. This chapter describes common strategies to identify dimerization of purinergic receptors (coimmunoprecipitation, bioluminescence resonance energy transfer (BRET), and immunoelectron microscopy) and to assess the alteration of their pharmacology (ligand binding, intracellular cAMP, and intracellular Ca(2+) assays). We have reported dimerization of purinergic receptors using these strategies in transfected human embryonic kidney 293T cells and native brain tissue. Our data suggest that homo- and hetero-oligomerization between purinergic receptors exert unique pharmacology in this receptor group. According to these discoveries, heterodimerization is likely to be employed for the "fine-tuning" of purinergic receptor signaling.

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function.

A(2A) adenosine receptor (A(2A)R), P2Y(1) receptor (P2Y(1)R) and P2Y(12) receptor (P2Y(12)R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca(2+) signaling evoked by the P2Y(1)R agonist, 2-methylthioladenosine 5' diphosphate (2MeSADP) was significantly inhibited by the A(2A)R antagonist (ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-α][1,3,5]triazin-5-yl amino]ethyl) phenol) and SCH442416) and the P2Y(12)R antagonist (ARC69931MX) (N6-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-ß,γ-dichloromethylene-ATP)) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y(1)R signaling by A(2A)R and P2Y(12)R antagonists was indeed mediated through A(2A)R and P2Y(12)R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.

Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A1 and P2Y2 receptors in rat brain.

BACKGROUND: Purines such as adenosine and ATP are now generally recognized as the regulators of many physiological functions, such as neurotransmission, pain, cardiac function, and immune responses. Purines exert their functions via purinergic receptors, which are divided into adenosine and P2 receptors. Recently, we demonstrated that the Gi/o-coupled adenosine A1 receptor (A1R) and Gq/11-coupled P2Y2 receptor (P2Y2R) form a heteromeric complex with unique pharmacology in co-transfected human embryonic kidney cells (HEK293T). However, the heteromeric interaction of A1R and P2Y2R in situ in brain is still largely unknown. FINDINGS: In the present study, we visualized the surface expression and co-localization of A1R and P2Y2R in both transfected HEK293T cells and in rat brain by confocal microscopy and more precisely by immunogold electron microscopy. Immunogold electron microscopy showed the evidence for the existence of homo- and hetero-dimers among A1R and P2Y2R at the neurons in cortex, cerebellum, and particularly cerebellar Purkinje cells, also supported by co-immunoprecipitation study. CONCLUSION: The results suggest that evidence for the existence of homo- and hetero-dimers of A1R and P2Y2R, not only in co-transfected cultured cells, but also in situ on the surface of neurons in various brain regions. While the homo-dimerization ratios displayed similar patterns in all three regions, the rates of hetero-dimerization were prominent in hippocampal pyramidal cells among the three regions.

Dimerization of G protein-coupled purinergic receptors: increasing the diversity of purinergic receptor signal responses and receptor functions.

It is well accepted that G protein-coupled receptors (GPCRs) arrange into dimers or higher-order oligomers that may modify various functions of GPCRs. GPCR-type purinergic receptors (i.e. adenosine and P2Y receptors) tend to form heterodimers with GPCRs not only of the different families but also of the same purinergic receptor families, leading to alterations in functional properties. In the present review, we focus on current knowledge of the formation of heterodimers between metabotropic purinergic receptors that activate novel functions in response to extracellular nucleosides/nucleotides, revealing that the dimerization seems to be employed for 'fine-tuning' of purinergic signaling. Thus, the relationship between adenosine and adenosine triphosphate is likely to be more and more intimate than simply being a metabolite of the other.

Ligand-induced rearrangements of the GABA(B) receptor revealed by fluorescence resonance energy transfer.

The gamma-aminobutyric acid type B receptor (GABA(B)R), one of the family C G-protein-coupled receptor members, exists as a heterodimer comprised of subunits GB1 and GB2. To clarify the ligand-induced activation mechanism of the GABA(B)R, each subunit was fused with either Cerulean or enhanced yellow fluorescent protein at its intracellular loop, and fluorescence resonance energy transfer (FRET) changes upon agonist application were monitored. As a result, FRET decreases were observed between GB1a loop 2 and GB2 loop 2 and between GB1a loop 2 and GB2 loop 1, suggesting the dissociation of intracellular domains during the receptor activation. Both intersubunit FRET pairs were expected to faithfully capture the activation of the original receptor as their pharmacological properties were highly similar to that of the wild-type receptor. However, the intrasubunit data suggest that the receptor activation does not involve major structural changes within the transmembrane domain of each subunit. By combining the results obtained from two different levels, it was concluded that the GABA(B)R activation by agonist is associated with an asymmetrical intersubunit rearrangement of GB1a and GB2 on the membrane. This type of activation mode, an intersubunit rearrangement without apparent intrahelical structural changes, appears commonly shared by the GABA(B)R and the metabotropic glutamate receptor 1alpha, another family C G-protein-coupled receptor previously studied by our group. Nevertheless, the directions of intracellular domain movements and its asymmetry observed here highlight the qualitative difference between the two receptors.

A highly conserved tryptophan residue in the fourth transmembrane domain of the A adenosine receptor is essential for ligand binding but not receptor homodimerization.

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A(1) adenosine receptor (A(1)R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A(1)R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A(1)R, as evident from coimmunoprecipitation and BRET(2) analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A(1)R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.

Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity for adenosine receptors.

Four bis-N-n-propyl analogues (3-6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and eudistomin D, a beta-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A(1), A(2A), and A(3) were examined. All the compounds (3-6) showed better potency as adenosine receptor ligands than caffeine. Bis-N-n-propylation (3 and 4, respectively) of the uracil ring in 1 and 2 resulted in higher affinity for A(1) and A(2A) adenosine receptors. Furthermore, it was found that a compound (5) possessing a n-propyloxy group at C-7 in compound 3 with a nitrogen at the beta-position of the pyridine ring (beta-N type) enhanced remarkably affinity for adenosine receptor A(3) subtype, while n-propyloxy substitution (compound 6) at C-5 in compound 4 with a nitrogen at the delta-position of the pyridine ring (delta-N type) reduced affinity for all the adenosine receptor, A(1), A(2A), and A(3). Among all the compounds (1-6) examined, compound 5 showed the most potent affinity for adenosine receptor A(3) subtype (K(i) value, 0.00382 microM).

Synthesis of eudistomin D analogues and its effects on adenosine receptors.

Six analogues (1-6) of eudistomin D, a beta-carboline alkaloid from a marine tunicate Eudistoma olivaceum, were synthesized, and their affinity and selectivity for adenosine receptors A(1), A(2A), and A(3) were examined. All the synthetic compounds 1-6 did not show affinity to the adenosine A(1) receptor. Delta-carboline 3 exhibited the most potent affinity to the adenosine receptor A(3) among compounds 1-6. Delta-carbolines 3 and 4 showed better affinity than the corresponding beta-carbolines 1 and 2, respectively, while N-methylation (2, 4, and 6, respectively) of the pyrrole ring in 1, 3, and 5 resulted in the reduced affinity to the adenosine A(3) receptor. On the other hand, an eudistomin D derivative, BED, exhibited modest affinity to all the receptors A(1), A(2A), and A(3) but no selectivity.

Synthesis of hybrid molecules of caffeine and eudistomin D and its effects on adenosine receptors.

Four hybrid molecules (1 and 12-14) of caffeine and eudistomin D, a beta-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A(1), A(2A), and A(3) were examined. It was found that all the compounds showed better potency as adenosine receptor ligands as compared with caffeine. Among them, a compound (13) possessing a nitrogen at the delta-position of the pyridine ring (delta-N type) showed the most potent affinity for adenosine receptor A(3) subtype, while N-methylation (14) of a pyrrole ring in 13 significantly lowered the potency as adenosine receptor ligands. Compounds (1 and 12) having a nitrogen at the beta-position of the pyridine ring (beta-N type) showed lower affinity than the corresponding delta-N type compounds (13 and 14), while compounds (10, 11, and 17) lacking a pyrrole ring between the pyridine and pyrimidine rings exhibited almost no affinity to the adenosine receptor subtypes examined.

Mitogen-activated protein kinase signaling is involved in suramin-induced neurite outgrowth in a neuronal cell line.

Suramin is a well-known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers. Previous study showed that suramin is an activator of extracellular signal-regulated kinase (ERK1/2) signaling in several cell lines including Chinese hamster ovary cells, although the physiological relevance of this activation remains uncertain. Here, it was shown that suramin enhances neurite outgrowth concomitant with activation of ERK1/2 in Neuro-2a cells, a neuronal cell line. These neurite outgrowth and ERK1/2 activation were significantly inhibited by PD98059, an inhibitor of mitogen-activated protein kinase kinase, as well as by activation of endogenous adenosine A2A receptors. The suramin-induced phosphorylation of ERK1/2 was also inhibited by inhibitors of Src family kinases. This attenuation of ERK1/2 activity was accompanied by a significant decrease in suramin-induced neurite outgrowth. These results suggest that suramin activates the Src/ERK1/2 signaling pathway that induces neurite outgrowth, both of which are negatively regulated by cAMP produced in response to activation of endogenous adenosine A2A receptors.

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor.

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).

Clathrin required for phosphorylation and internalization of beta2-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2).

Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the beta2-adrenergic receptor (beta2AR). However, the interaction of beta2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of beta2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis.

Functions of heteromeric association between adenosine and P2Y receptors.

It is now well accepted that G protein-coupled receptors (GPCRs) can be directly associated, as either homo- or hetero-oligomers, to alter their functions. G protein-coupled purinergic receptors, classified as adenosine receptors, and P2Y receptors (ATP receptors) are also found to oligomerize each other to alter their pharmacology. Specifically, adenosine receptor of A1 subtype (A1R) is able to form a heteromeric complex with P2Y receptor of P2Y1 type (P2Y1R) either in heterologously transfected cells or in rat brain tissues, as demonstrated by coimmunoprecipitation or bioluminescence resonance energy transfer methods in addition to double immunocytochemistry. It is shown that the heteromerization between A1R and P2Y1R generates an adenosine receptor with P2Y-like agonistic pharmacology, i.e., a potent P2Y1R agonist, adenosine 5'-O-(2-thiodiphosphate), binds the A1R binding pocket of the A1R/P2Y1R complex and inhibits adenylyl cyclase activity via Gi/o protein. This hetero-oligomerization between adenosine receptor and P2Y receptor might be one of the mechanisms for the adenine nucleotide-mediated inhibition of neurotransmitter release. The oligomerization of purinergic receptors is thus considered as an important regulation system in the central nervous system.

Identification of specific [3H]adenine-binding sites in rat brain membranes.

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.

Functional expression of single-chain heterodimeric G-protein-coupled receptors for adenosine and dopamine.

The direct homo- and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A(2A)R) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A(2A)R/A(2A)R vs. A(2A)R/D2R). In order to stimulate the heteromerization of A(2A)R and D2R, we have designed a single-polypeptide-chain heterodimeric A(2A)R/D2R complex by fusing the C-terminus of the A(2A)R via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A(2A)R/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.