Jean Lucas-Lenard

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

Abstract: The human cannabinoid receptor CB1 functionally couples primarily to Gi‐, but also to Gs‐mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl‐terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist‐independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs‐ and not a Gi‐mediated pathway. Treatment with the CB1‐specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP‐55,940 to the L341A/A342L receptor was not markedly different from that for the wild‐type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Ligand binding and modulation of cyclic AMP levels depend on the chemical nature of residue 192 of the human cannabinoid receptor 1.

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein‐coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild‐type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP‐55,940 and the aminoalkylindole WIN 55,212‐2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP‐55,940, whereas WIN 55,212‐2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand‐induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212‐2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP‐55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212‐2 binding, the data suggest a role for Lys192 in WIN 55,212‐2‐induced receptor activation.

Cell killing by viruses. IV. Cell killing and protein synthesis inhibition by vesicular stomatitis virus require the same gene functions.

Abstract Temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus representing the five known complementation groups of the Indiana serotype were used to determine which viral gene functions were required for the expression of (i) cell-killing (CK) particle activity, and (ii) the inhibition of host cell protein synthesis. Cell-killing particle activity was quantitated by analyzing single-cell survival curves generated by infecting the Vero line of green monkey kidney cells at low multiplicities (mpfp ≤ 5) and at permissive (30°) and nonpermissive (40°) temperatures. Cellular protein synthesis inhibition was measured by difference analysis of polyacrylamide-gel-electropherogram patterns obtained from uninfected mouse L cells and cells infected at high multiplicity (mpfp = 100–500) at these two temperatures. Analyses of cell killing and protein synthesis inhibition by is mutants at nonpermissive temperature demonstrate that both of these attributes require the same viral gene functions and that they are conjointly expressed or not expressed, tsckp+,psi+ or tsckp−,psi− , respectively. Complementation groups I and IV were subdivided into tsckp+,psi+ and tsckp−,psi− mutants, establishing that the viral proteins required for the expression of cell killing and protein synthesis inhibition need not be fully functional. Considering these and previous results [Virology 57: 321 1974; 63: 176 1975; 69: 378 1976] we conclude that, although the expression of both cell killing and protein synthesis inhibition by VS virus does not require infectious virus, it does depend upon transcription of viral genes N and NS by virion-associated transcriptase and their translation into minimally functional proteins. We postulate that viral proteins N and NS then interact with minimally functional L protein to produce the putative proximate or ultimate factor(s) directly responsible for cell killing and protein synthesis inhibition by VS virus. The lack of cellular protein synthesis inhibition by several is mutants following challenge at high multiplicities (mpfp = 500) at 40° casts some doubt on the interpretation of experiments that appear to support assignment of these functions to preformed viral proteins.

Inhibition of mouse L cell protein synthesis by ultraviolet-irradiated vesicular stomatitis virus requires viral transcription.

Abstract Ultraviolet-irradiated vesicular stomatitis (VS) virus (Indiana serotype) was used to determine which viral gene functions are required for expression of cellular protein synthesis inhibition. To carry out this experiment a method, described in this report, was employed that allows the simultaneous quantitation of viral and host protein synthesis. We demonstrated earlier using ultraviolet irradiation that to express cell killing, only about one-fifth of the VS virus genome corresponding to proteins N and NS need be transcribed. Previous results using temperature-sensitive mutants of VS virus indicated that inhibition of protein synthesis and cell killing are conjointly expressed. In this report it is demonstrated that the inhibition of cellular protein synthesis by a high multiplicity ( m pfp = 100) of uv-irradiated VS virus, like cell killing, requires the transcription and translation of N (and possibly NS) protein.

Lack of Correlation Between the Accumulation of Plus-Strand Leader RNA and the Inhibition of Protein and RNA Synthesis in Vesicular Stomatitis Virus Infected Mouse L Cells

The inhibition of protein synthesis in mouse L cells infected by vesicular stomatitis virus (VSV) requires expression of two regions (one large and one small) of the viral genome, as determined by target size analysis. The inhibition of host RNA synthesis was also shown to be dependent on expression of two regions of the VSV genome, most likely the same ones. In some cases, such as in cells infected by mutants T1026R1, or tsG41 at 40 degrees, or moderately uv irradiated VSV, only one of the two regions was expressed, yet cellular protein and RNA synthesis was decreased. This suggests that the product of each region of the viral genome can act independently. In these instances the severity of the inhibition was dependent on both the length of the infection period and the multiplicity of infection. The identity of neither gene product is known, but it has been suggested that small product is plus-strand leader RNA. As shown herein, however, there was no correlation between the extent of host macromolecular synthesis inhibition and the quantity of leader RNA in infected cells.

Further studies on the inhibition of cellular protein synthesis by vesicular stomatitis virus

Abstract After infection of L cells by vesicular stomatitis virus, the rate of protein synthesis steadily declines. Simultaneously, polysomes disaggregate and 80 S ribosomes accumulate. Transit time studies indicate that the average rate of polypeptide chain elongation is virtually identical for infected and uninfected cells. Cellular mRNAs remain intact and potentially functional in infected cells, as determined by their ability to be translated into cellular proteins in nuclease-treated reticulocyte lysates. More template activity is present in infected cells than in uninfected cells as a result of the presence of viral mRNAs in infected cells. Extracts from infected L cells translate their endogenous mRNAs less efficiently than do comparable extracts from uninfected cells. Nuclease-treated extracts from infected cells also show a lesser ability to translate given amounts of exogenous L cell or vesicular stomatitis virus mRNAs than do comparable extracts from uninfected cells. This observation suggests that the translation of both cellular and viral mRNAs is affected equally by the lesion induced in the cell by virus infection. The lesion appears to act at the level of initiation of protein synthesis and results in an underutilization of the total template activity present in infected cells.

Inhibition of Cellular Protein Synthesis After Virus Infection

This review is concerned with the effects of picornavirus infection on the biosynthesis of macromolecules in animal cells. Soon after infection of animal cells with a member of the picornavirus group, there is an inhibition of cellular RNA and protein synthesis. Later in infection, the synthesis of cellular DNA is also decreased. During this time, viral constituents are synthesized and assembly of viral progeny occurs. Late in infection, there is an irreversible loss of the capacity of the cell to synthesize any macromolecules, host or viral, and finally, the cell dies.

Induction of thymidylate synthetase activity in Tetrahymena by cyclic guanosine monophosphate.

The induction of TMP synthetase activity in Tetrahymena pyriformis depended upon growth conditions. Enzymatic activity was low in cells grown in complex medium, and was high in cells grown in, or shifted to, defined medium. TMP synthetase activity rose 5 hours after the shift from complex to defined medium using uracil as the pyrimidine source. The time of induction was decreased to 312 hours using dUMP as the pyrimidine source. cGMP or its dibutyryl derivative, but not cAMP, caused the induction of TMP synthetase activity in cells grown in complex medium. Caffeine, but not theophylline, mimicked the cGMP response. cAMP decreased both the cGMP and caffeine mediated increases in TMP synthetase activity. This is the first demonstration of an effect of cGMP on induction of an enzyme of pyrimidine metabolism in any cellular system.

2. Protein Synthesis—Peptide Chain Elongation

Publisher Summary This chapter discusses the process of peptide chain elongation. For studies on the mechanism of polypeptide chain elongation extensive use has been made of the model system in which polyuridylic acid, poly(U), directs the formation of polyphenylalanyl-tRNA. The requirements for this in vitro system are high salt washed ribosomes, Phe-tRNA, poly (U), guanosine triphosphate, Mg 2+ , and NH 4 + ions, a sulfhydryl reagent such as dithiothreitol and three protein factors which are present in the supernatant fraction of lysed cells. In peptide chain elongation, Protein growth is accomplished by a cyclic process involving aminoacyl-tRNA binding, peptidyl transfer, peptidyl tRNA translocation, and exposure of a new triplet codon through movement of the ribosome on mRNA. These factors have been recently designated as elongation factor thermo unstable (EF-Tu), elongation factor thermo stable (EF-Ts), and elongation factor G (EF-G), the new symbols being intended to replace the various designations used for the factors in different laboratories. When isolated from the soluble fraction of the cell, EF-Tu and EF-Ts are associated, and this complex is referred to as EF-T. The respective factors from various bacterial species may be interchanged in the partial reactions of peptide chain elongation as well as in the overall polymerization reaction. The amount of EF-T and EF-G in the bacterial cell is a significant percentage of the total soluble proteins. The EF-G content of Escherichia coli cells has been estimated under different growth conditions to be 2-3 % and 6% and the EF-T content as 2% and 3%. The relative amount of each factor compared to ribosomes remains constant at different growth rates, suggesting that the synthesis of the polypeptide chain elongation factors is coordinated with that of ribosomes.

The Influence of the Host Cell on the Inhibition of Virus Protein Synthesis in Cells Doubly Infected with Vesicular Stomatitis Virus and Mengovirus

The ability of mengovirus to inhibit the synthesis of vesicular stomatitis virus (VSV) proteins and of VSV to inhibit the synthesis of mengovirus proteins during double infection in three different cell lines was investigated. Although cellular protein synthesis was inhibited after infection of cells by each virus, the ability of one virus to decrease translation of the mRNA species of the co-infecting virus varied with the cell type. Superinfection of mengovirus-infected L-929 cells by VSV resulted in essentially no inhibition in the synthesis of either mengovirus or VSV proteins. In HeLa cells and CHO cells the synthesis of both VSV and mengovirus proteins was inhibited under conditions of simultaneous or sequential infection. The inhibition of VSV protein synthesis after infection of HeLa cells by mengovirus was not a result of a modification or inactivation of virus mRNAs. When extracted from double infected cells, the VSV mRNAs manifested normal biological activity, as determined by their ability to stimulate the synthesis of VSV proteins in a micrococcal nuclease-treated cell-free system from L cells. The interference of non-interference of one virus by another in different cell lines was also measured by quantifying the number of infectious particles produced in each cell line. The results were similar to those reported above for protein synthesis inhibition. These experiments suggest that the interference of mengovirus with VSV mRNA translation in HeLa cells is not necessarily reflective of the mechanism by which mengovirus inhibits cellular protein synthesis. Also, the host cell appears to influence the extent or nature of the interference of one virus by the other.