Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and ß-Arrestins 2.

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or ß-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/ß-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gß1 or NOP/ß-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects.

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.

Completion of molecular characterization of Toscana phlebovirus genome: nucleotide sequence, coding strategy of M genomic segment and its amino acid sequence comparison to other phleboviruses.

The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Unkuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and GC glycoproteins were placed at residues 297 and 936 of the precursor.

Organization of the M genomic segment of Toscana phlebovirus.

The nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3' NSm-G1 -G2 5'

Toscana virus genomic L segment: molecular cloning, coding strategy and amino acid sequence in comparison with other negative strand RNA viruses.

The complete nucleotide sequence of Toscana (TOS) virus (Bunyaviridae, Phlebovirus) L segment was determined. The L segment is 6404 nucleotides long, containing a single open reading frame (ORF) in the viral complementary sense coding for a protein of 2095 amino acids that, as in the case of negative strand RNA viruses, could be part of the RNA polymerase of TOS virus. This ORF is expressed by a messenger RNA (mRNA) as long as the genomic segment. Like the mRNAs expressed by the genomic segments of the other Bunyaviruses, the L mRNA has non-templated sequences at the 5' end. The comparison of TOS L protein sequence with the corresponding sequences of other negative strand RNA viruses showed a very high homology only with the Rift Valley Fever (RVF) virus. The residues conserved between the two proteins are mainly concentrated in the central region and contain three DD motifs proposed by Argos (1988) to be functional domains of DNA and RNA polymerases. The complete sequence of the Toscana virus L genomic segment has been deposited in the EMBL library with the accession number X68414.

Analysis of 3' and 5' ends of N and NSs messenger RNAs of Toscana Phlebovirus.

The 5' and 3' ends of N and NSs mRNAs, transcribed from the S segment of Toscana Phlebovirus, were analyzed by oligonucleotide primer extension and S1 nuclease mapping procedures. The results showed that both mRNAs acquired, at their 5' end, approximately 9-15 nucleotides not present in the viral template, suggesting an initiation transcription mechanism similar to the one described for influenza virus. Furthermore, the 3' ends of the two mRNAs were located in a sequence motif conserved in the S segment of two other Phleboviruses, the Rift Valley Fever and Sandfly Fever Sicilian viruses. This finding suggests the possible involvement of this sequence in the mechanism of transcription termination.

Sequences and coding strategies of the S RNAs of Toscana and Rift Valley fever viruses compared to those of Punta Toro, Sicilian Sandfly fever, and Uukuniemi viruses.

The sequences and coding strategies of the S RNAs of two viruses, Toscana (TOS) and the M12 derivative of Rift Valley fever ZH-548 (RVF, Phlebovirus genus, Bunyaviridae) have been determined from cDNA clones and compared to the previously published sequences of Punta Toro (PT), Sandfly fever Sicilian (SFS), and Uukuniemi (UUK) viruses. All five viruses exhibit an ambisense coding strategy for their small (S) RNA species, i.e., one gene product (the NSs protein) is encoded in the 5' half of the viral RNA, a second (the N protein) is encoded in the sequence complementary to the 3' half. The terminal nucleotides of the S RNAs of the five viruses are comparable through 13-14 residues. The 3' and 5' ends of these S RNAs have inverted complementary compositions. Three phleboviruses (TOS, SFS, and RVFV) exhibit comparable G-rich, centrally located intergenic sequences, albeit of different lengths. These sequences have a number of similar motifs at, or immediately following, the end of the coding regions, motifs that may be involved in their S mRNA transcription termination processes. The other two viruses (UUK, PT) have AT-rich intergenic sequences that have the potential to form secondary structure. They lack the G-rich sequences or particular sequence motifs recognized in the other three virus RNAs. The deduced sizes of the TOS and RVFV N proteins are 27,704 and 27,430 kDa (respectively). Their NSS proteins are 36,677 and 29,903 kDa (respectively). When aligned, the deduced sequences of the N proteins of the five viruses exhibit homologies ranging from 54 to 30%. The order of homology to RVFV N protein is PT greater than TOS greater than SFS greater than UUK; to TOS N protein it is PT greater than or equal to RVF greater than SFS greater than UUK. The sequences of the NSS proteins are less similar, with values ranging from 30 to less than 17%. The order of homology to RVFV NSS is SFS greater than PT greater than TOS greater than UUK. Due to these more distant relationships, the homologies to TOS NSS protein are less clear.