RGS19 enhances cell proliferation through its C-terminal PDZ motif.

Regulator of G protein signaling 19 (RGS19), also known as Galpha-interacting protein (GAIP), is a GTPase activating protein (GAP) for Galpha(i) subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.

Activation of STAT3 by specific Galpha subunits and multiple Gbetagamma dimers.

The hematopoietic-specific G(q) subfamily members, Galpha(16) and Galpha(14) proteins have recently been shown to be capable of stimulating the signal transducer and activator of transcription 3 (STAT3) as well as STAT1. In the present study we examined whether this activation was STAT-member specific as well as determining the possible involvement of Gbetagamma dimers. Despite clear stimulation of STAT3, the constitutively active mutants of Galpha(16) (Galpha(16)QL) and Galpha(14) (Galpha(14)QL) failed to induce the phosphorylation of several STAT family members, including STAT2, STAT4 and STAT5 in human embryonic kidney 293 cells. On the other hand, transient expression of specific combinations of Gbetagamma complexes induced STAT3 phosphorylation. Among the 48 combinations tested, 13 permutations of Gbetagamma stimulated STAT3 phosphorylation and all of them contain the neuronal-specific Ggamma(2), Ggamma(4), Ggamma(7) and Ggamma(9). These results suggested that the activation of STAT family members by Galpha(16) or Galpha(14) was selective and that distinct combinations of Gbetagamma complexes can also regulate the STAT signaling pathway.

Gbeta3 forms distinct dimers with specific Ggamma subunits and preferentially activates the beta3 isoform of phospholipase C.

Heterotrimeric G proteins regulate multiple effectors of which some are mediated via the Gbetagamma dimers. There is evidence to suggest that the functions of Gbetagamma dimers are not shared by all possible permutations of Gbetagamma complexes. Here, we report our efforts in defining the formation of distinct Gbetagamma dimers and their functional differences in activating phospholipase Cbeta (PLCbeta) isoforms. Co-immunoprecipitation assays using Cos-7 cells transiently expressing 48 different combinations of Gbeta(1-4) and Ggamma(1-5, 7-13) subunits showed that Gbeta(1) and Gbeta(4) could form dimers with all known Ggamma subunits, whereas several dimers could not be observed for Gbeta(2) and Gbeta(3). All Gbeta(1)gamma and Gbeta(2)gamma dimers significantly stimulated PLCbeta isoforms (PLCbeta(2)> or =PLCbeta(3)>PLCbeta(1)), but Gbeta(3)gamma and Gbeta(4)gamma dimers were poor activators of PLCbeta(1) and exhibited preference for PLCbeta(3) and PLCbeta(2), respectively. All Gbeta subunits revealed to date contain the previously identified PLCbeta(2)-interacting residues, but their neighboring residues in the proposed 3-D structures are different. To test if differences in these neighboring residues affect the interactions with PLCbeta isoforms, we generated several Gbeta(3) mutants by replacing one or more of these residues with their Gbeta(1) counterparts. One of these mutants (M120I, S140A and A141G triple mutant) acquired enhanced PLCbeta(2)-activating functions when co-expressed with different Ggamma subunits, while the corresponding stimulation on PLCbeta(3) was not altered. Taken together, our results show that the exact composition of a Gbetagamma dimer can determine its selectivity for activating PLCbeta isoforms, and certain residues in Gbeta(3) may account for the preferential stimulation of PLCbeta(3) by Gbeta(3)gamma dimers.

Dopaminergic and adrenergic toxicities on SK-N-MC human neuroblastoma cells are mediated through G protein signaling and oxidative stress.

Dopamine and norepinephrine are neurotransmitters which participate in various regulatory functions of the human brain. These functions are lost in neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. In this study, we used SK-N-MC neuroblastoma cells to investigate the cytotoxicities of high concentrations of dopamine and norepinephrine on neuronal cells. Dopamine, norepinephrine, as well as their corresponding synthetic agonists (SKF38393 and isoproterenol, respectively) triggered SK-N-MC cell death when applied at 50-100 muM persistently for 2 days. This catecholamine-induced cell death appears to be neuronal specific, as demonstrated by their inabilities of triggering apoptosis of A549 lung carcinoma cells and Cos-7 kidney fibroblasts. By pretreating SK-N-MC cells with target-specific inhibitors before administration of catecholamine, components of G protein signaling (i.e. G( s )/cAMP/PKA), monoamine oxidases, nitric oxide synthase, c-Jun N-terminal kinase and oxidative stress were found to be involved in this dopamine/norepinephrine-induced cytotoxicity, which subsequently led to caspase-dependent and -independent apoptotic responses as well as DNA degradation. In contrast, agonists of G( i )-coupled dopamine receptors and adrenergic receptors (quinpirole and UK14,304, respectively) were incapable of triggering apoptosis of SK-N-MC cells. Our results suggest that both G protein (G( s ))-mediated signaling cascade and oxidative stress participate in the dopamine/norepinephrine-induced neuronal apoptosis.