Two RGS proteins that inhibit Galpha(o) and Galpha(q) signaling in C. elegans neurons require a Gbeta(5)-like subunit for function.

BACKGROUND: Gbeta proteins have traditionally been thought to complex with Ggamma proteins to function as subunits of G protein heterotrimers. The divergent Gbeta(5) protein, however, can bind either Ggamma proteins or regulator of G protein signaling (RGS) proteins that contain a G gamma-like (GGL) domain. RGS proteins inhibit G protein signaling by acting as Galpha GTPase activators. While Gbeta(5) appears to bind RGS proteins in vivo, its association with Ggamma proteins in vivo has not been clearly demonstrated. It is unclear how Gbeta(5) might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit Galpha(o) and Galpha(q) signaling, respectively. RESULTS: We knocked out the gene encoding the C. elegans Gbeta(5) ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable, and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. CONCLUSIONS: Gbeta(5) functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gbeta(5), these RGS proteins have little or no function. The formation of RGS-Gbeta(5) complexes is required for the expression or stability of both the RGS and Gbeta(5) proteins. Appropriate RGS-Gbeta(5) complexes regulate both Galpha(o) and Galpha(q) proteins in vivo.

Multiple RGS proteins alter neural G protein signaling to allow C. elegans to rapidly change behavior when fed.

Regulators of G protein signaling (RGS proteins) inhibit heterotrimeric G protein signaling by activating G protein GTPase activity. Many mammalian RGS proteins are expressed in the brain and can act in vitro on the neural G protein G(o), but the biological purpose of this multiplicity of regulators is not clear. We have analyzed all 13 RGS genes in Caenorhabditis elegans and found that three of them influence the aspect of egg-laying behavior controlled by G(o) signaling. A previously studied RGS protein, EGL-10, affects egg laying under all conditions tested. The other two RGS proteins, RGS-1 and RGS-2, act as G(o) GTPase activators in vitro but, unlike EGL-10, they do not strongly affect egg laying when worms are allowed to feed constantly. However, rgs-1; rgs-2 double mutants fail to rapidly induce egg-laying behavior when refed after starvation. Thus EGL-10 sets baseline levels of signaling, while RGS-1 and RGS-2 appear to redundantly alter signaling to cause appropriate behavioral responses to food.

Antagonism between G(o)alpha and G(q)alpha in Caenorhabditis elegans: the RGS protein EAT-16 is necessary for G(o)alpha signaling and regulates G(q)alpha activity.

To elucidate the cellular role of the heterotrimeric G protein G(o), we have taken a molecular genetic approach in Caenorhabditis elegans. We screened for suppressors of activated GOA-1 (G(o)alpha) that do not simply decrease its expression and found mutations in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G(o)alpha and negatively regulate EGL-30 (G(q)alpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G(q)/G(11) in COS-7 cells. Animals defective in both sag-1 and eat-16 are inviable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G(q)alpha activity. Analysis of these mutations indicates that the G(o) and G(q) pathways function antagonistically in C. elegans, and that G(o)alpha negatively regulates the G(q) pathway, possibly via EAT-16 or SAG-1. We propose that a major cellular role of G(o) is to antagonize signaling by G(q).

A new family of G-protein regulators - the RGS proteins.

Genetic experiments have recently been used to identify a family of 'regulator of G-protein signaling' (RGS) proteins, which downregulate signaling by heterotrimeric G proteins. The first biochemical studies of RGS proteins have shown that they accelerate the GTPase activities of G-protein alpha subunits, thus driving G proteins into their inactive GDP-bound forms. The physiological significance of the large number of different RGS proteins remains to be explored.

EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins.

The frequencies of certain periodic behaviors of the nematode C. elegans are regulated in a dose-dependent manner by the activity of the gene egl-10. These behaviors are modulated oppositely by the activity of the G protein alpha subunit gene goa-1, suggesting that egl-10 may regulate a G protein signaling pathway in a dose-dependent fashion. egl-10 encodes a protein similar to Sst2p, a negative regulator of G protein signaling in yeast. EGL-10 protein is localized in neural processes, where it may function in neurotransmitter signaling. Two previously known and 13 newly identified mammalian genes have similarity to egl-10 and SST2, and we propose that members of this family regulate many G protein signaling pathways.

DHR3: a Drosophila steroid receptor homolog.

In Drosophila the steroid hormone ecdysone triggers a genetic regulatory hierarchy in which ecdysone combines with a receptor protein to form a complex that induces the transcription of a small class of "early" genes, which encode transcription factors that regulate other genes. We previously reported that one of the early genes, E75, encodes members of the steroid receptor superfamily. Using an E75 hybridization probe, we have identified two additional Drosophila genes that encode members of this superfamily. One of these is the ecdysone receptor gene, EcR, as previously reported. In this work, we examine the sequence, genomic organization, and developmental expression of the other gene, DHR3, which, like E75, encodes one of a growing number of "orphan" receptors for which ligands have not yet been identified. The structure of the DHR3 protein is strikingly similar to that of the MHR3 protein (e.g., 97% amino acid identity for the DNA binding domains), another orphan receptor encoded by an ecdysone-inducible early gene of another insect, Manduca sexta. The temporal developmental profile for DHR3 expression closely parallels that for the ecdysone titer and for the ecdysone-inducible E75 and E74 Drosophila early genes. The structural similarity to a Manduca early gene and the expression similarities to Drosophila early genes suggest that the DHR3 gene may also belong to the early gene class.

The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily.

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.

In-vivo epidermal nuclear reactions: a selective process.

Epidermal in-vivo nuclear reactions of IgG occur primarily in patients with mixed connective tissue disease or systemic lupus erythematosus and have been associated with high titres of circulating antibodies to ribonucleoprotein (RNP). This study was carried out to examine whether these epidermal nuclear reactions are true or simply an excision artefact. We observed the epidermal nuclear reactions for IgG only and not for other immunoglobulins in both in-vivo and in-vitro organ-culture studies, despite the presence of antinuclear antibodies (ANA) of all immunoglobulin classes. The association of the in-vitro epidermal nuclear reactions with serum RNP antibodies, although not absolute was statistically significant. The absorption of the serum with extractable nuclear antigen (ENA) preparation diminished the nuclear reactivity on tissue explants. In addition, the penetration of ANA into the nuclei of skin explants was both time and temperature dependent and was inhibited by sodium azide and by oligomycin. We conclude that the epidermal nuclear staining reactions observed by direct immunofluorescence on skin biopsies is selective and that the penetration of IgG into the epidermal cell nuclei is an active process and not an artefact.

Visualization and quantification of transmural concentration profiles of macromolecules across the arterial wall.

Transport parameters that describe a macromolecule entering the arterial wall from plasma can be obtained from concentration profiles of the labeled macromolecule entering the tissue. A new technique has been developed for measuring such concentration profiles, which offers spatial resolution superior to methods that measure profiles of radiolabeled macromolecules by serially sectioning tissue in planes parallel to the endothelium. In addition, this new method preserves cellular organization and tissue structure and permits measurement of concentration profiles underlying focal endothelial injuries or vascular lesions. The technique quantifies the concentration of a protein by measuring associated peroxidase activity. Although the present study was performed using horseradish peroxidase (HRP), the same principles can be applied to other macromolecules linked to HRP or microperoxidase. The colored reaction product of HRP was detected in transverse aortic sections using an image processing system. In the present study, profiles obtained by this new method were validated by comparison with HRP concentration profiles in rat aortas obtained by a serial slicing technique using radiolabeled HRP. We used the technique to measure high-resolution HRP concentration profiles in the intima and media of normal animals. These concentration profiles suggest that the internal elastic lamina acts as a major barrier to transport of macromolecules across the wall of the normal rat aorta. The new method should allow concentration profiles for macromolecules to be quantified in tissue surrounding vessels in the microcirculation, within the thickened intima of large vessels, and across coronary artery walls.

Scl-70 antigen stability and its effect on antibody detection in scleroderma.

We examined 38 patients with scleroderma, 10 with systemic lupus and 10 normal subjects for Scl-70 antibodies by the gel precipitation and by the immunoblot methods. Increased incidence of Scl-70 antibodies in scleroderma were found by the immunoblot method (55%) compared to the gel precipitation methods (40 or 42% depending on the test kits used). Immunoblot analysis of the antigen prepared with 4.0 M NaCl extraction of rabbit thymus acetone powder revealed antigen to be 100, 86, 80 and 70 kDa. However, mainly a single band of 70 kDa was obtained upon extraction of rabbit thymus with 0.3 M NaCl and of calf thymus antigen. Our data support the suggestion that different molecular species of the Scl-70 antigen have variable binding affinities to nuclear DNA. We suggest that the presence of various molecular forms of the antigen may be a result of this differential binding affinity to DNA and the partial proteolytic digestion of 100 kDa from the antigen as reported by others.

A standardized method of detecting antibodies to extractable nuclear antigens (RNP and Sm) by gel precipitation.

Because of the importance of the detection of antibodies to RNP and Sm in the diagnosis of mixed connective tissue disease, systemic lupus erythematosus and certain forms of systemic sclerosis, the various factors which influence the sensitivity of the gel precipitation method for the detection of these antibodies were investigated. The agarose concentration, thickness, well sizes and distance between wells influence the sensitivity of the precipitin reactions. Suitable conditions for a sensitive and reproducible test system are specified. Because of variations in the antigenic composition of extractable nuclear antigen preparations made by different methods and from different tissue sources, a two-dimensional "chessboard titration" using serial twofold dilutions of a reference serum to test doubling dilutions of antigen preparations is recommended for standardizing different lots of antigen. Using the above-mentioned standardized agarose plates and appropriate reference serum with anti-Sm and/or anti-RNP antibodies in a chessboard titration, it is recommended that acceptable use dilution of antigen preparation be two doubling dilutions before reaching an endpoint. The reference sera selected should be compared to US reference standards supplied by the Center for Disease Control.

Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory.