Regulators of G Protein Signaling and Transient Activation of Signaling: EXPERIMENTAL AND COMPUTATIONAL ANALYSIS REVEALS NEGATIVE AND POSITIVE FEEDBACK CONTROLS ON G PROTEIN ACTIVITY

Cellular responses to hormones and neurotransmitters are necessarily transient. The mating pheromone signal in yeast is typical. Signal initiation requires cell surface receptors, a G protein heterotrimer, and down-stream effectors. Signal inactivation requires Sst2, a regulator of G protein signaling (RGS) protein that accelerates GTPase activity. We conducted a quantitative analysis of RGS and G protein expression and devised computational models that describe their activity in vivo. These results indicated that pheromone-dependent transcriptional induction of the RGS protein constitutes a negative feedback loop that leads to desensitization. Modeling also suggested the presence of a positive feedback loop leading to resensitization of the pathway. In confirmation of the model, we found that the RGS protein is ubiquitinated and degraded in response to pheromone stimulation. We identified and quantitated these positive and negative feedback loops, which account for the transient response to external signals observed in vivo

β 2 -Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling

Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity

A series of mathematical models was used to quantitatively characterize pheromone-stimulated kinase activation and determine how mitogen-activated protein (MAP) kinase specificity is achieved. The findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity

Feedback Regulation in the Lactose Operon: A Mathematical Modeling Study and Comparison with Experimental Data

A mathematical model for the regulation of induction in the lac operon in Escherichia coli is presented. This model takes into account the dynamics of the permease facilitating the internalization of external lactose; internal lactose; β-galactosidase, which is involved in the conversion of lactose to allolactose, glucose and galactose; the allolactose interactions with the lac repressor; and mRNA. The final model consists of five nonlinear differential delay equations with delays due to the transcription and translation process. We have paid particular attention to the estimation of the parameters in the model. We have tested our model against two sets of β-galactosidase activity versus time data, as well as a set of data on β-galactosidase activity during periodic phosphate feeding. In all three cases we find excellent agreement between the data and the model predictions. Analytical and numerical studies also indicate that for physiologically realistic values of the external lactose and the bacterial growth rate, a regime exists where there may be bistable steady-state behavior, and that this corresponds to a cusp bifurcation in the model dynamics

Biological modelling / Biomodélisation Modeling operon dynamics: the tryptophan and lactose operons as paradigms

Abstract Understanding the regulation of gene control networks and their ensuing dynamics will be a critical component in the understanding of the mountain of genomic data being currently collected. This paper reviews recent mathematical modeling work on the tryptophan and lactose operons which are, respectively, the classical paradigms for repressible and inducible operons

Primary ovarian leiomyoma: A case report

INTRODUCTION: Primary ovarian leiomyoma is a rare benign tumour of the ovary seen in women between 20 and 65 years old. It is usually diagnosed incidentally during pelvic examination or pathologic examination after surgery. PRESENTATION OF CASE: We describe a case of unilateral, ovarian leiomyoma. Transvaginal ultrasonography and magnetic resonance imaging (MRI) revealed a right adnexial mass. Unilateral salpingo-oophorectomy was performed, and histological examination revealed a leiomyoma arising primarily in the ovary. The diagnosis was confirmed immunohistochemically. DISCUSSION: The tumour may be asymptomatic or may manifest with lower abdominal pain like in our case. The definitive diagnosis of these lesions is difficult prior to surgical removal. Because there is no pathognomonic symptoms or characteristic imaging findings. The correct diagnosis of an ovarian leiomyoma requires identification of the smooth muscle nature of the tumour. CONCLUSION: This rare tumour of the ovary should be considered in the differential diagnosis of solid ovarian masses. An immunohistochemical analysis is recommended for definitive diagnosis