Differentially Regulated Expression of Endogenous RGS4 and RGS7

Abstract

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27-28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30 -40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short halflife. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.G proteins transduce signals across the plasma membrane by sequential interactions with cell surface receptors and appropriate second messenger-producing effectors (e.g. enzymes and ion channels). These interactions are modulated by nucleotide-driven conformational changes in the ␣ subunits of heterotrimeric G proteins (G␣).1 A ligand-bound receptor catalyzes the exchange of GDP for GTP on its cognate G␣ and the dissociation of G␣ from the complex of G protein ␤ and ␥ subunits (G␤␥). These dissociated subunits are competent to modulate the activity of effectors. The duration of G protein-mediated responses are dependent on the intrinsic GTPase rate of G␣ and on extrinsic factors, such as regulators of G protein signaling (RGS proteins).RGS proteins serve to regulate G protein signaling by functioning as GTPase-activating proteins (GAPs). GAP activity can sharpen the termination of a signal upon removal of a stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network ...

A Novel Mechanism Involving Coordinated Regulation of Nuclear Levels and Acetylation of NF-YA and Bcl6 Activates RGS4 Transcription

“…We focused on the RGS4 gene in view of its association with a number of pathologies and the important role of neurally enriched RGS4 in G protein signaling. Acute increases in RGS4 transcripts in confluent PC6 cells (15) presented an excellent experimental system to elucidate how the RGS4 gene is activated in a neuronal-like cell. First, we identified and cloned the human RGS4 promoter.…”

“…Studies have shown that several RGS genes are induced under certain physiological conditions, for example RGS1 during mitogenic activation of lymphocytes (9), RGS2 in the early stage of 3T3-L1 differentiation to adipocytes (10), RGS16 during genotoxic stress (11), and the yeast RGS gene SST2 by pheromone (12). Regulation of RGS4 expression has also been noted in facial motoneuronal precursors during embryonic development in mice (13), in epithelial and endothelial cells during tubulogenesis (14), and in rat PC12 cells during cell confluence (15). In the study by Grillet et al (13), in situ hybridization was used to demonstrate that RGS4 transcription is increased in differentiating and post-mitotic neurons in the developing mouse nervous system.…”

“…Signaling-RGS4, a tightly regulated GTPase-activating protein (13)(14)(15)23), plays a critical role as a negative modulator of G protein-coupled receptor signaling via G␣ i or G␣ q (3,4,49,50) in brain and other tissues. Fig.…”

“…The LAP isoform of rat C/EBP␤ was amplified from rat genomic DNA and subcloned in pcDNA3 vector (Invitrogen). RGS4-specific antibody (U1079) (15) was generously provided by Dr. Susan Mumby. Oligonucleotide primers and other molecular biological reagents were obtained from the University of Iowa DNA Core Facility.…”

RGS9–2 Negatively Modulates l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia in Experimental Parkinson's Disease

“…Western blot analysis was performed as previously described Krumins et al, 2004) using the following primary antibodies: rabbit antiserum against the C terminus of RGS2 (CKKPQITTEPHAT; 1:1000; a generous gift from D. Siderovski, University of North Carolina, Chapel Hill, NC); rabbit antisera against rat RGS4 (Krumins et al, 2004) (1:2000; a generous gift from S. Mumby, University of Texas Southwestern Medical Center, Dallas, TX), affinity purified anti-RGS7 antibodies (Upstate Biotechnologies; 1:10,000), protein A-purified rabbit antibodies made in our laboratory to histagged RGS9c bacterially expressed fragment (He et al, 1998), and SGS rabbit antisera against G␤5 (Zhang et al, 1996) (1:5000; a generous gift from W. Simonds, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). The specificities of these RGS and G␤5 antibodies were further confirmed using brain extracts from wild-type (wt) and mutant mice (supplemental Fig.…”