A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models.

Steroidogenic acute regulatory protein (StAR) mediates translocation of cholesterol to the inner membranes of steroidogenic mitochondria, where it serves as a substrate for steroid synthesis. Transcription of StAR in the gonads and adrenal cells is upregulated by trophic hormones, involves downstream signaling pathways and a cohort of trans-factors acting as activators or suppressors of StAR transcription. This study suggests that a 21 basepair long sequence positioned at -81/-61 of the murine StAR promoter is sufficient to confer a robust hormonal activation of transcription in ovarian granulosa cells treated with FSH. We show that recombinant GATA-4 and CCAAT/enhancer-binding protein beta (C/EBPbeta) bind to the promoter at -66/-61 and -81/-70 and activate transcription of a reporter gene when co-expressed in heterologous human embryonic kidney 293 (HEK293) cells. In this cell model, C/EBPbeta and GATA-4 synergize in a sequence dependent manner and p300/CBP further maximizes their joint activities. Inhibitors of the transcriptional activators, such as liver-enriched inhibiting protein (C/EBPbeta-LIP), Friend of GATA-4 (FOG-2) protein and the viral E1A protein abolished the respective factor-dependent activities in HEK293 cells. Binding assays suggest that a dual binding of C/EBPbeta and GATA-4 to the promoter depends on the molar ratio of the factors present while demonstrating GATA-4 predominant association with the promoter DNA. This pattern may reflect on StAR expression at the time of corpus luteum formation when C/EBPbeta levels peak, as does StAR expression.

Mature-onset obesity in interleukin-1 receptor I knockout mice.

Interleukin-1 (IL-1) is a major mediator of inflammation that exerts its biological activities through the IL-1 type I receptor (IL-1RI). The body weights of IL-1RI(-/-) mice of both sexes started to deviate from those of wild-type mice at 5-6 months of age and were 20% higher at 9 months of age. Visceral and subcutaneous fat mass, measured by dual-energy X-ray absorptiometry and magnetic resonance imaging, was markedly (1.5- to 2.5-fold) increased. Lean body mass and crown-rump length were also slightly (11 and 5%, respectively) increased, as was serum IGF-I. Obese IL-1RI(-/-) mice were insulin resistant, as evidenced by hyperinsulinemia, decreased glucose tolerance, and insulin sensitivity. To elucidate the mechanisms for the development of obesity, young pre-obese IL-1RI(-/-) mice were investigated. They showed decreased suppression of body weight and food intake in response to systemic leptin treatment. The decreased leptin responsiveness was even more pronounced in older obese animals. Moreover, spontaneous locomotor activity and fat utilization, as measured by respiratory quotient, were decreased in pre-obese IL-1RI(-/-) mice. In conclusion, lack of IL-1RI-mediated biological activity causes mature-onset obesity. This obese phenotype is preceded by decreased leptin sensitivity, fat utilization, and locomotor activity.

Combination of two activating mutations in one HOG1 gene forms hyperactive enzymes that induce growth arrest.

Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.

Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological activity.

Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity.