Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes.

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.

Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family.

A general property of signal transduction pathways is that prolonged stimulation decreases responsiveness, a phenomenon termed desensitization. Yeast cells stimulated with mating pheromone activate a heterotrimeric G-protein-linked, MAP-kinase-dependent signalling pathway that induces G1-phase cell-cycle arrest and morphological differentiation (reviewed in refs 1, 2). Eventually the cells desensitize to pheromone and resume growth. Genetic studies have demonstrated the relative importance of a desensitization mechanism that uses the SST2 gene product, Sst2p. Here we identify a mammalian gene family termed RGS (for regulator of G-protein signalling) that encodes structural and functional homologues of Sst2p. Introduction of RGS family members into yeast blunts signal transduction through the pheromone-response pathway. Like SST2 (refs 8-10), they negatively regulate this pathway at a point upstream or at the level of the G protein. The RGS family members also markedly impair MAP kinase activation by mammalian G-protein-linked receptors, indicating the existence and importance of an SST2-like desensitization mechanism in mammalian cells.

Requirement for the expression of poly(ADP-ribose) polymerase during the early stages of differentiation of 3T3-L1 preadipocytes, as studied by antisense RNA induction.

Poly(ADP-ribose) polymerase (PADPRP) is biologically significant in the rejoining of DNA strand breaks. Post confluent cultures of 3T3-L1 preadipocytes showed marked increases in PADPRP protein and activity when the cells were induced to differentiate into adipocytes. When this increase in PADPRP expression was prevented in stably transfected 3T3-L1 cells by induction of PADPRP antisense RNA synthesis, the cells did not differentiate nor undergo the two or three rounds of DNA replication that are required for initiation of the differentiation process. 3T3-L1 cells expressing PADPRP antisense RNA under differentiation conditions were easily detached from plates and in some cases eventually died. When newly expressed PADPRP protein and DNA synthesis was assessed in cells at zero time or at 24 h after induction of differentiation by incorporation of bromodeoxyuridine or [3H]thymidine into DNA, significant incorporation was shown to occur in control cells after 24 h, but not in antisense cells. Furthermore, during the first 24 h, the co-immunoprecipitation of PADPRP and DNA polymerase alpha was observed in control cells, whereas no such complex formation was noted in the induced antisense cells, nor in uninduced control cells.

Depletion of poly(ADP-ribose) polymerase by antisense RNA expression results in a delay in DNA strand break rejoining.

The effects of inducible expression of poly(ADP-ribose) polymerase (PADPRP) antisense RNA in HeLa cells were determined in order to gain further insight into the biological roles of the poly(ADP-ribosyl)ation modification of nuclear proteins. A recombinant expression plasmid was prepared with the mouse mammary tumor virus (MMTV) promoter upstream of the antisense-oriented PADPRP cDNA. Expression of the antisense RNA was under strict control, with negligible effects on cell growth being apparent in the absence of inducer. Consistent with the previously described stability of PADPRP (half-life of at least 2 days, in vivo), 48-72 h were required after induction of antisense RNA expression by dexamethasone for the abundant concentration of PADPRP, normally present in HeLa cells, to be reduced by greater than 80%. The depletion of endogenous PADPRP as mediated by induced antisense RNA expression was established by: (i) a progressive synthesis of antisense transcripts in cells as assessed by Northern analysis; (ii) an 80% decrease in activity of the enzyme; and (iii) a greater than 90% reduction in the cellular content of PADPRP protein, as demonstrated by both immunoblotting and immunohistochemical analysis in intact cells. Several biological parameters were monitored in cells depleted of PADPRP. The chromatin of PADPRP-depleted cells was shown to have an altered structure as assessed by deoxyribonuclease I susceptibility. Cell morphology was also altered, with multinucleated aggregates being evident 72 h after induction of antisense RNA expression. Cells depleted of PADPRP were not able to commence DNA strand break joining of damaged DNA. However, DNA repair capacity was re-established at later time periods, indicating that PADPRP may contribute to alterations in chromatin structure that occur initially in DNA strand break rejoining and that the concentration of the enzyme in nuclei exceeds the requirement for DNA repair/replication.

Cell cycle regulation of an exogenous human poly(ADP-ribose) polymerase cDNA introduced into murine cells.

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.