Mammalian RGS proteins: barbarians at the gate.
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Amino Acid Sequence↗
Biomedical subjects
D M Berman
Publications and source records attributed to D M Berman.
Regional differences in adrenoceptor binding and fat cell lipolysis in obese, postmenopausal women.
In women there is an increase in visceral obesity, subcutaneous abdominal adipocyte lipolysis, and risk of cardiovascular disease (CVD) associated with weight gain after menopause. The mechanisms underlying this increase in adrenoreceptor (AR)-agonist catecholamine-stimulated lipolysis and abdominal obesity in postmenopausal women were studied in intact adipocytes isolated from the abdominal and gluteal subcutaneous fat depots in 19 obese (48% +/- 1% body fat, mean +/- SE) women with a mean +/- SE age of 58 +/- 1 years. The fat cell size and adipose tissue lipoprotein lipase (ATLPL) activity were similar in both sites. The maximal lipolytic responsiveness and sensitivity to isoproterenol were higher (P < .05) in abdominal compared with gluteal adipocytes, but maximal lipolytic response to a post-AR agent was similar. Abdominal adipocytes had a higher beta-AR ([3H]-CGP-12177) and alpha2-AR ([3H]-yohimbine) affinity than gluteal cells (P < .05), lower alpha2-AR density (P < .05), but similar beta-AR density as gluteal cells. Both abdominal and gluteal cell size correlated with alpha2-AR density (P < .01), but not with beta-AR density. Thus, a higher beta-AR affinity and lower alpha2-AR relative to beta-AR density may explain the higher in vitro catecholamine-mediated lipolysis in abdominal compared with gluteal adipocytes in obese, postmenopausal women.
Adipocytes↗
Activation of transducin guanosine triphosphatase by two proteins of the RGS family.
RGS proteins (regulators of G protein signaling) constitute a newly appreciated group of negative regulators of G protein signaling. Several members of this group stimulate the guanosine triphosphatase (GTPase) activity of various G protein alpha-subunits, including the photoreceptor G protein, transducin. In photoreceptor cells transducin GTPase is known to be substantially accelerated by the coordinated action of the gamma-subunit of its effector enzyme, cGMP phosphodiesterase (PDE gamma), and another yet unidentified membrane-associated protein factor. Here we test the possibility that this factor belongs to the RGS family of GTPase stimulators. We report a detailed kinetic analysis of transducin GTPase activation by two members of the RGS family, RGS4 and G alpha interacting protein (GAIP). RGS4, being at least 5-fold more potent than GAIP, stimulates the rate of transducin GTPase by 2 orders of magnitude. Neither RGS4 nor GAIP requires PDE gamma for activating transducin. Rather, PDE gamma causes a partial reversal of transducin GTPase activation by RGS proteins. The effect of PDE gamma is based on a decreased apparent affinity of RGS for the alpha-subunit of transducin. Our observations indicate that GTPase activity of transducin can be activated by at least two distinct mechanisms, one based on the action of RGS proteins alone and another involving the cooperative action of the effector enzyme and another protein.
3',5'-Cyclic-GMP Phosphodiesterases↗
Structure of RGS4 bound to AlF4--activated G(i alpha1): stabilization of the transition state for GTP hydrolysis.
RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 A resolution crystal structure of the RGS protein RGS4 complexed with G(i alpha1)-Mg2+-GDP-AlF4 . Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(i alpha1), but does not contribute catalytic residues that directly interact with either GDP or AlF4-. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(i alpha1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G(i alpha1) is also consistent with the activity of RGS proteins as antagonists of G(alpha) effectors.
Aluminum Compounds↗
RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha.
RGS proteins constitute a newly appreciated and large group of negative regulators of G protein signaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the Gi alpha subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for Gq alpha, the G alpha protein responsible for activation of phospholipase C beta. Furthermore, these RGS proteins block activation of phospholipase C beta by guanosine 5'-(3-O-thio) triphosphate-Gq alpha. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G alpha for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.
Adenylyl Cyclases↗
The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis.
RGS proteins constitute a newly appreciated group of negative regulators of G protein signaling. Discovered by genetic screens in yeast, worms, and other organisms, two mammalian RGS proteins, RGS4 and GAIP, act as GTPase-activating proteins for members of the Gi family of G protein alpha subunits. We have purified recombinant RGS4 to homogeneity and demonstrate that it acts catalytically to stimulate GTP hydrolysis by Gi proteins. Furthermore, RGS4 stabilizes the transition state for GTP hydrolysis, as evidenced by its high affinity for the GDP-AlF4--bound forms of Goalpha and Gialpha and its relatively low affinity for the GTPgammaS- and GDP-bound forms of these proteins. Consequently, RGS4 is most likely not a downstream effector for activated Galpha subunits. All members of the Gi subfamily of proteins tested are substrates for RGS4 (including Gtalpha and Gzalpha); the protein has lower affinity for Gqalpha, and it does not stimulate the GTPase activity of Gsalpha or G12alpha.
Aluminum Compounds↗
GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits.
A novel class of regulators of G protein signaling (RGS) proteins has been identified recently. Genetic evidence suggests that RGS proteins inhibit G protein-mediated signaling at the level of the receptor-G protein interaction or the G protein alpha subunit itself. We have found that two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by Gi alpha 1 at least 40-fold. All Gi subfamily members assayed were substrates for these GAPs; Gs alpha was not. RGS4 activates the GTPase activity of certain Gi alpha 1 mutants (e.g., R178C), but not others (e.g., Q204L). The GAP activity of RGS proteins is consistent with their proposed role as negative regulators of G protein-mediated signaling.
Base Sequence↗
Expression and regulation of steroid 5 alpha-reductase in the urogenital tract of the fetal rat.
Two androgens, testosterone and dihydrotestosterone, are required for the development of the male urogenital tract in the rat. Testosterone is secreted by the fetal testes and is thought to elicit differentiation of the Wolffian ducts into seminal vesicles, vas deferens, and epididymides. Testosterone is converted into dihydrotestosterone by steroid 5 alpha-reductase in the urogenital tract, and this conversion is necessary for the differentiation of the prostate and external genitalia. Genes encoding two 5 alpha-reductase isozymes, designated type 1 and type 2, have been identified. We examined the expression and regulation of these genes on days 17-21 in the urogenital tracts of male and female fetuses. Expression of the type 1 gene predominated in epithelial cells, whereas type 2 gene expression was limited to mesenchymal cells. Surprisingly, this expression pattern was detected in both testosterone-dependent and dihydrotestosterone-dependent anlagen of the urogenital tract and was the same in both male and female fetuses. Furthermore, transcripts encoding the two isozymes were present in their respective cell types before the overt differentiation of internal genitalia. Androgens stimulated expression of the type 2 gene in the urogenital tracts of both sexes, but did not effect expression of the type 1 gene or the cell type-specific expression patterns of the 5 alpha-reductase genes. In the adult prostate, 5 alpha-reductase gene expression is under feedforward control, in which the product of the enzyme, dihydrotestosterone, stimulates the expression of the gene. However, no evidence for feedforward regulation of either 5 alpha-reductase gene was detected in the fetus.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Changes in membrane potential associated with cell swelling and regulatory volume decrease in barnacle muscle cells.
Our aim was to test the effect of hypotonicity and extracellular Ca2+ (Cao) on cell volume and membrane potential (VM) in barnacle muscle cells. Under isotonic conditions the resting VM of isolated cells mounted in the experimental chamber exposed to either Ca(2+)-free or Ca(2+)-containing (11 mM) solutions was -46.3 +/- 1.0 mV (n = 24) and -56.2 +/- 0.9 mV (n = 38), respectively. In the absence of Cao, the cells depolarized at a rate of 2.3 +/- 0.47 mV/hr; the presence of Cao reduced this rate of depolarization by 2.9-fold. Both in the absence or presence of Cao, the cells swelled in response to hypotonicity but underwent regulatory volume decrease (RVD) when Cao was present. Addition of the Ca2+ channel blocker, verapamil (0.1 mM), inhibited the Cao-dependent RVD. The percentage of cells responding with RVD increased with larger hypotonic challenges. There was a Cao-independent direct relationship between cell swelling and membrane depolarization which can be explained by dilution of the concentration of intracellular K+ ([K+]i). RVD was accompanied by a small hyperpolarization (3.0 +/- 0.38 mV/2 hr) which may represent increases in [K+]i during cell shrinking and activation of a conductive pathway. The results indicate the following: (1) the presence of Cao stabilizes VM; (2) cell swelling produces a depolarization which can be explained by dilution of [K+]i; (3) cell swelling activates a verapamil-sensitive Ca2+ influx responsible for promoting RVD; and (4) RVD is accompanied by a hyperpolarization which may result from activation of a conductive pathway.
Animals↗
The molecular genetics of steroid 5 alpha-reductases.
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3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Ca2+ stores in smooth muscle cells: Ca2+ buffering and coupling to AVP-evoked inositol phosphate synthesis.
Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while decreasing the Ca2+ content of sarcoplasmic reticulum (SR) stores in cultured A7r5 cells at rest and after activation with 8-arginine vasopressin (AVP). Decreasing Ca2+ influx by reducing extracellular Ca2+ or by treatment with verapamil had no effect on resting [Ca2+]cyt but significantly inhibited the AVP-evoked Ca2+ transients (delta Ca2+). Neither treatment affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Likewise, basal [3H]InsP were unaffected by brief (10-30 s) exposures to thapsigargin (TG), while AVP-induced [3H]InsP synthesis was significantly augmented. Similar treatment with TG rapidly increased resting [Ca2+]cyt and decreased SR Ca2+ by 9-25% as manifested by decreased delta Ca2+. By contrast, ryanodine induced slow increases in [Ca2+]cyt that stabilized within 30 min; subsequent AVP-induced delta Ca2+ were attenuated by 50%. Ryanodine had no effect on either basal or stimulated [3H]InsP levels. Agents that elevate adenosine 3',5'-cyclic monophosphate (cAMP) such as caffeine, 8-bromo-cAMP, and forskolin inhibited AVP-evoked [3H]InsP formation. These observations provide further characterization of a communication pathway between the AVP-sensitive Ca2+ stores in the SR and the plasmalemmal enzyme system involved in the synthesis of inositol 1,4,5-trisphosphate. This pathway is manifested by an inverse relationship between the Ca2+ content of an AVP-sensitive, ryanodine-insensitive SR Ca2+ store and evoked [3H]InsP synthesis and may represent an important component in the tonic regulation of resting [Ca2+]cyt and vasoconstrictor- and hormone-evoked SR Ca2+ release.
Arginine Vasopressin↗
Atrial natriuretic peptide inhibited the natriferic and hydrosmotic effects of arginine vasotocin on toad skin.
The effect of rat atrial natriuretic peptide (rANP) on hormonal stimulated osmotic water permeability (Jw, hydrosmotic effect) and net ion transport (short-circuit current, SCC, natriferic effect) was studied on toad skin, a tissue with functional similarities to the mammalian distal nephron, in order to assess actions on transport mechanisms. Rat atrial natriuretic peptide, rANP-99-126 (rANP) inhibited stimulated SCC and Jw to submaximal concentrations of arginine vasotocin (AVT) at a site before cyclic AMP generation. The angiotensin-converting enzyme inhibitor (ACEI) MK-422 did not modify the inhibitory effect of ANP in the stimulated Jw.
Animals↗
Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes.
The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.
Animals↗
Effects of angiotensin I of the American bullfrog Rana catesbeiana on amphibian tissues.
1. The effect of bullfrog angiotensin I [Asp1, Val5, Asn9] angiotensin I, (AT I) on short-circuit current (SCC) on isolated toad skin and aorta contractility was examined. 2. AT I increased SCC in toad skin, the effect was partially inhibited by angiotensin-converting enzyme inhibitor (ACEI) teprotide. 3. AT I induced contractile responses in isolated rings of toad aorta. This effect was partially inhibited by captopril and completely blocked by the peptide antagonist [Sar1, Ile8] angiotensin II. 4. Present results indicate that this homologue AT I would act in amphibian tissues by conversion to AT II.
Angiotensin I↗
External Ca effect on water permeability, regulatory volume decrease, and extracellular space in barnacle muscle cells.
The effect of extracellular Ca2+ (Cao) on sarcolemmal hydraulic water permeability (L'p), regulatory volume decrease (RVD), and extracellular space (ECS) was studied in barnacle muscle cells. Absence or presence of Cao had no effect on L'p [0 Cao = 2.762 +/- 0.098 x 10(-5), and 11 mM Cao = 2.720 +/- 0.222 x 10(-5) cm.kg.s-1 x osmol x 1-kgH2O-1]. Likewise, cells exposed to anisosmotic media (for < 30 min) behaved as osmometers in 0 and 11 mM Cao, showing similar slopes and intercepts in van't Hoff plots. At longer incubation times, however, hyposmotic conditions promoted a Cao-dependent RVD. The relationship between Cao and the percentage of cells responding with RVD to a hyposmotic challenge was sigmoidal (half-maximal Cao = 4.83 mM). The mean rate of RVD (40 nl/min) was independent of the level of swelling in response to hyposmotic challenges. However, the magnitude of RVD increased with larger hyposmotic challenges. Both the presence of Cao and hypotonicity reduced the "apparent" ECS by 47 +/- 6 and 39 +/- 6%, respectively. Three-dimensional reconstruction of autoradiographs of the cells was made to interpret these results.
Animals↗
Stored calcium modulates inositol phosphate synthesis in cultured smooth muscle cells.
Cytosolic Ca2+ concentrations ([Ca2+]cyt) and [3H]inositol phosphates ([3H]InsP) were correlated while varying the Ca2+ content of the sarcoplasmic reticulum (SR) in cultured A7r5 cells at rest and during activation with [Arg8]-vasopressin (AVP). Thapsigargin (TG) raised and superfusion with 0 Ca2+ lowered [Ca2+]cyt, but both treatments decreased SR Ca2+ and AVP-evoked Ca2+ transients. Neither TG nor 0 Ca2+ affected basal [3H]InsP, but both treatments increased AVP-evoked synthesis of [3H]InsP. Exposure for several minutes to 40 mM K+ solution, BAY K 8644, or low-Na+ solution all elevated [Ca2+]cyt and, thereby, increased SR Ca2+, as manifested by augmented AVP-evoked Ca2+ transients. In all three cases, AVP-evoked, but not basal, [3H]InsP were reduced. The inhibitory effect of 40 mM K+ on AVP-evoked [3H]InsP synthesis was blocked when SR Ca2+ uptake was prevented by TG. Brief (30-s) exposures to 40 mM K+, which elevated [Ca2+]cyt but not SR Ca2+ loading, did not modify AVP-evoked [3H]InsP synthesis or Ca2+ transients. These results demonstrate an inverse relationship between SR Ca2+ content and evoked [3H]-InsP synthesis. Moreover, they suggest that SR Ca2+ may serve as a signal that modulates sarcolemmal [3H]InsP formation.
Arginine Vasopressin↗
On the mechanism of vasodilating action of berberine: possible role of inositol lipid signaling system.
We have studied the effect of the alkaloid berberine on the contraction of guinea pig aortic strips induced by various stimuli. Berberine (25-200 microM) inhibited the response of the strips to norepinephrine and histamine, but did not decrease the high K(+)-elicited contraction. The antagonism of berberine was not competitive because in the presence of the alkaloid, maximum response to agonists could not be obtained. Analysis of the drug's effect on the time course of norepinephrine-induced contraction showed that berberine reduced both the rate and the relative contribution to developed tension of the initial, rapid phase, whereas the slow, later component was less affected. Berberine inhibited the response of aortic strips incubated in 0 mM Ca++ to norepinephrine, but did not reduce caffeine-induced contraction and also inhibited phospholipase C-activated contractile response, which has been ascribed to production of inositol phosphate-3 in smooth muscle cells. In cultured arterial smooth muscle cells (A7r5 line), the alkaloid did not significantly decrease the production of inositol phosphates activated by Arg8-vasopressin. The pattern of berberine action is difficult to reconcile with an involvement of the contractile machinery and suggests that the drug has no effect on the voltage-operated calcium channels. Although an antagonism at the receptors or an increase of cyclic AMP or cyclic GMP cannot be completely excluded, we suggest that at least one component of the berberine inhibitory effect may be due to its action on some step of the chain of events linking receptors to contractile response.
Animals↗
Deletion of steroid 5 alpha-reductase 2 gene in male pseudohermaphroditism.
The conversion of testosterone into dihydrotestosterone by steroid 5 alpha-reductase is a key reaction in androgen action, and is essential both for the formation of the male phenotype during embryogenesis and for androgen-mediated growth of tissues such as the prostate. Single gene defects that impair this conversion lead to pseudohermaphroditism in which 46X,Y males have male internal urogenital tracts, but female external genitalia. We have described the isolation of a human 5 alpha-reductase complementary DNA from prostate. Subsequent cloning and genetic studies showed that this gene (designated 5 alpha-reductase 1) was normal in patients with 5 alpha-reductase deficiency. We report here the isolation of a second 5 alpha-reductase cDNA by expression cloning and the polymerase chain reaction. The biochemical and pharmacological properties of this cDNA-encoded enzyme (designated 5 alpha-reductase 2) are consistent with it being the major isozyme in genital tissue. A deletion in this gene is present in two related individuals with male pseudohermaphroditism caused by 5 alpha-reductase deficiency. These results verify the existence of at least two 5 alpha-reductases in man and provide insight into a fundamental hormone-mediated event in male sexual differentiation.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗