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2.
Mol Cell Biol ; 21(6): 1921-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11238928

ABSTRACT

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.


Subject(s)
Cell Differentiation/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Protein Serine-Threonine Kinases , Thyroid Gland/cytology , Thyroid Gland/metabolism , rap1 GTP-Binding Proteins/metabolism , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Genes, ras , Mutation , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Wistar , Thyroid Gland/drug effects , Thyrotropin/metabolism , Thyrotropin/pharmacology , rap1 GTP-Binding Proteins/genetics
3.
J Biol Chem ; 275(45): 34901-8, 2000 Nov 10.
Article in English | MEDLINE | ID: mdl-10934204

ABSTRACT

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.


Subject(s)
DNA-Binding Proteins , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Nerve Tissue Proteins , Transcription Factors , rap1 GTP-Binding Proteins/metabolism , ras Guanine Nucleotide Exchange Factors/metabolism , Amino Acid Sequence , Blotting, Northern , Brain/metabolism , Calcium/metabolism , Catalytic Domain , Cell Line , DNA, Complementary/metabolism , Diglycerides/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Humans , Molecular Sequence Data , Mutagenesis , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Homology, Amino Acid , Tissue Distribution , Tumor Cells, Cultured , ets-Domain Protein Elk-1 , rap GTP-Binding Proteins/metabolism , ras Guanine Nucleotide Exchange Factors/chemistry
4.
J Biol Chem ; 275(18): 13406-10, 2000 May 05.
Article in English | MEDLINE | ID: mdl-10747847

ABSTRACT

Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.


Subject(s)
Guanine Nucleotide Exchange Factors/genetics , ral GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , Guanine Nucleotide Exchange Factors/isolation & purification , Guanine Nucleotide Exchange Factors/metabolism , Molecular Sequence Data , Sequence Alignment , Sequence Analysis
5.
Hypertension ; 34(4 Pt 1): 631-7, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10523338

ABSTRACT

Renin and aldosterone secretion is often lower in blacks than in whites, characteristics that resemble a milder form of Liddle syndrome in which a mutation in the amiloride-sensitive epithelial sodium channel (ENaC) of the kidney results in enhanced resorption of sodium. In the present study, we looked for evidence that the intrinsic level of ENaC activity is indeed higher in blacks than in whites. In overnight urine samples collected from young people (249 white and 181 black subjects, mean age 13.4 years), the urinary aldosterone/potassium ratio, which is typically very low in Liddle syndrome, was lower in blacks than in whites: 0.421+/-0.024 (mean+/-SE) versus 0.582+/-0.016 nmol/mmol (P<0.0001). In addition, all but 1 of 5 molecular variants in ENaC were much more common in blacks than in whites. G442V in the beta-subunit, present in 16% of the blacks and in only 1 white, was associated with parameters reflective of a greater Na retention and potentially a higher ENaC activity: a lower plasma aldosterone concentration (P=0.070), a lower urinary aldosterone excretion rate (P=0.052), a higher potassium excretion rate (P=0.048), and a lower urinary aldosterone/potassium ratio (P=0.027). In a second cohort consisting of 126 black and 161 white normotensive subjects and 232 black and 188 white hypertensive subjects, betaG442V did not show a significant association with hypertension (P=0.089). On the other hand, a variant that was twice as common in whites, alphaT663A, was associated with being normotensive both in blacks (P=0.018) and in whites (P=0.034). Expression of either betaG442V or alphaT663A in Xenopus oocytes did not result in a change in basal Na current, consistent with the variants being in linkage disequilibrium with alleles at active loci. In conclusion, several lines of evidence are presented to suggest that ENaC activity is higher in blacks than in whites, which could contribute to racial differences in Na retention and the risk for hypertension.


Subject(s)
Aldosterone/metabolism , Hypertension/genetics , Potassium/metabolism , Sodium Channels/genetics , Sodium Channels/metabolism , Adolescent , Aldosterone/blood , Aldosterone/urine , Black People/genetics , Blood Pressure/genetics , Cohort Studies , Epithelial Cells/metabolism , Exons , Female , Humans , Male , Potassium/blood , Potassium/urine , Renin/metabolism , Risk Factors , White People/genetics
6.
Hypertension ; 34(2): 315-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10454460

ABSTRACT

Blacks appear, on average, to retain more Na than whites. A higher production rate of mineralocorticoids could explain the greater Na retention in blacks. Although production of aldosterone has been shown to be lower in blacks, the level of another mineralocorticoid may be increased. Plasma levels of deoxycorticosterone and cortisol were measured in young whites (n=23; age=16.4+/-3.1[SD] years) and young blacks (n=25; age=13.8+/-1.3 years). Blacks had lower plasma levels of renin activity and aldosterone and lower urinary aldosterone excretion rates; thus, they appeared to be representative of blacks that retain additional Na. Plasma deoxycorticosterone levels were lower in blacks than in whites both at baseline (247+/-161 versus 381+/-270 pmol/L, P=0.048) and after stimulation with adrenocorticotropic hormone (822+/-294 versus 1127+/-628 pmol/L at 30 minutes, P=0.047; 925+/-366 versus 1440+/-834 pmol/L at 60 minutes, P=0.013). Cortisol levels were also lower in blacks at baseline (P=0.014) but were not significantly different from levels in whites after stimulation with adrenocorticotropic hormone. In a larger cohort of 407 whites (age=12.0+/-2.9 years) and 247 blacks (age=12.9+/-3.1 years), 18-hydroxycortisol excretion rates were also lower in blacks (P=0. 021). In conclusion, increased Na retention in blacks does not appear to be secondary to increased production of either aldosterone, deoxycorticosterone, cortisol, or 18-hydroxycortisol. A primary renal mechanism may mediate the increase in Na reabsorption in blacks.


Subject(s)
Black People , Mineralocorticoids/blood , White People , Adolescent , Adult , Age Factors , Aldosterone/blood , Aldosterone/urine , Cohort Studies , Data Interpretation, Statistical , Desoxycorticosterone/blood , Female , Humans , Hydrocortisone/analogs & derivatives , Hydrocortisone/blood , Hydrocortisone/urine , Male , Radioimmunoassay , Renin/blood , Sodium/metabolism
7.
J Clin Invest ; 103(5): 667-73, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10074483

ABSTRACT

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.


Subject(s)
Calcium-Binding Proteins/metabolism , Hypertension/genetics , Hypertension/metabolism , Ligases , Sodium Channels/metabolism , Ubiquitin-Protein Ligases , Animals , Calcium-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport , Epithelial Cells/metabolism , Mutation , Nedd4 Ubiquitin Protein Ligases , Rats , Sodium Channels/genetics , Syndrome , Xenopus , Xenopus Proteins
8.
DNA Seq ; 9(5-6): 295-306, 1998.
Article in English | MEDLINE | ID: mdl-10524756

ABSTRACT

The tryptophan-bounded WW domains ofNedd4 bind to the proline-tyrosine (PY) motifs contained in the C-terminal cytoplasmic region of the beta and gamma subunits of the rat amiloride-sensitive sodium channel (ENaC). In patients with Liddle's syndrome, the PY motif is mutated and the channel remains constitutively activated leading to sodium retention and hypertension. Although the function ofNedd4 is unknown, it contains a highly conserved ubiquitin protein ligase domain that may attach ubiquitin to ENaC, targeting it for degradation or it may modulate ENaC activity through another undetermined pathway. Xenopus laevis-derived cells, such as oocytes and the A6 kidney cell line, are important models currently used for the study of ENaC regulation. We describe the X. laevis homologue of Nedd4 (xNedd4). A partial clone, approximately 2.6 Kb, was isolated from an aldosterone-treated A6 cell cDNA library. Further 5' sequence, approximately 1.2 Kb, was obtained using a modified 5' rapid amplification of cDNA (RACE) protocol and cDNA from untreated A6 cells as the substrate. The identity and similarity of xNedd4 with human Nedd4 are approximately 63 and 71%, respectively. xNedd4 contains the C2, ubiquitin protein ligase, and 4 WW domains previously described for Nedd4 from other species.


Subject(s)
Xenopus laevis/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Ligases/chemistry , Molecular Sequence Data , Sequence Homology, Amino Acid , Ubiquitin-Protein Ligases
9.
J Anim Sci ; 64(2): 433-43, 1987 Feb.
Article in English | MEDLINE | ID: mdl-3558149

ABSTRACT

The present study was undertaken to determine the relationship between dose of porcine growth hormone (pGH) and growth performance of pigs. Porcine GH was administered daily for 35 d [buffer-injected control = (C); 10 micrograms/kg body weight (BW) = (L); 30 micrograms/kg BW = (M); 70 micrograms/kg BW = (H)] to barrows (initial wt = 50 kg). Growth rate was significantly increased by pGH (14% for H dose vs C). Feed efficiency was increased in a dose-related manner (L = 7%, M = 10%, H = 17%) by pGH. There was a concurrent change in carcass composition of pGH-treated pigs. The H dose of pGH decreased the percentage of carcass lipid by 25% (P less than .05). Muscle mass was significantly increased in H vs C pigs (31 vs 26 kg). Serum insulin-like growth factor 1 (IGF-1) concentration increased in a manner that was linearly related to the pGH dose (r = .87). No antibodies to pGH were detected in any of the pigs. In summary, these results extend our earlier findings that pGH increases growth performance markedly. Based on the present findings it appears that the maximally effective dose of pGH is greater than 70 micrograms.kg BW-1.d-1 since several indices of the growth-promoting and metabolic effects of pGH (% carcass protein, % carcass lipid and feed efficiency) had not plateaued.


Subject(s)
Body Weight/drug effects , Growth Hormone/pharmacology , Swine/growth & development , Animals , Dose-Response Relationship, Drug , Growth Hormone/administration & dosage , Injections, Intramuscular , Male , Orchiectomy
10.
J Anim Sci ; 63(5): 1389-99, 1986 Nov.
Article in English | MEDLINE | ID: mdl-3098726

ABSTRACT

The current study was undertaken to determine the effects of human growth hormone-releasing factor [hpGRF-(1-44)-NH2] on growth performance in pigs and whether this response was comparable to exogenous porcine growth hormone (pGH) treatment. Preliminary studies were conducted to determine if GRF increased plasma GH concentration after iv and im injection and the nature of the dose response. Growth hormone-releasing factor stimulated the release of pGH in a dose-dependent fashion, although the individual responses varied widely among pigs. The results from the im study were used to determine the dose of GRF to use for a 30-d growth trial. Thirty-six Yorkshire-Duroc barrows (initial wt 50 kg) were randomly allotted to one of three experimental groups (C = control, GRF and pGH). Pigs were treated daily with 30 micrograms of GRF/kg body weight by im injection in the neck. Pigs treated with pGH were also given 30 micrograms/kg body weight by im injection. Growth rate was increased 10% by pGH vs C pigs (P less than .05). Growth rate was not affected by GRF; however, hot and chilled carcass weights were increased 5% vs C pigs (P less than .05). On an absolute basis, adipose tissue mass was unaffected by pGH or GRF. Carcass lipid (percent of soft-tissue mass) was decreased 13% by GRF (P less than .05) and 18% by pGH (P less than .05). Muscle mass was significantly increased by pGH but not by GRF. There was a trend for feed efficiency to be improved by GRF; however, this was not different from control pigs. In contrast, pGH increased feed efficiency 19% vs control pigs (P less than .05). Chronic administration of GRF increased anterior pituitary weight but did not affect pituitary GH content or concentration. When blood was taken 3 h post-injection, both GRF- and pGH-treated pigs had lower blood-urea nitrogen concentrations. Serum glucose was significantly elevated by both GRF and pGH treatment. This was associated with an elevation in serum insulin. These results indicate that increasing the GH concentration in blood by either exogenous GH or GRF enhances growth performance. The effects of pGH were more marked than for GRF. Further studies are needed to determine the optimal dose of GRF to administer in growth trials and the appropriate pattern of GRF administration in order to determine whether GRF will enhance pig growth performance to the extent that exogenous pGH does.


Subject(s)
Growth Hormone-Releasing Hormone/pharmacology , Growth Hormone/pharmacology , Swine/growth & development , Animals , Body Composition/drug effects , Body Weight/drug effects , Dose-Response Relationship, Drug , Growth Hormone/blood , Male
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