Elucidation of Mechanisms Modulating the Conformation and Function of β-Arrestins by G Protein-Coupled Receptors

Arrestins are cytosolic G protein-coupled receptor (GPCR) binding proteins that regulate several facets of GPCR signaling. Once bound to agonist-occupied receptors, arrestins recruit elements of the clathrin-dependent endocytic machinery, resulting in removal of GPCRs from the plasma membrane. The fate of internalized receptors is determined by the stability of the GPCR-arrestin complex, which is itself dictated by several factors, including ligand structure, receptor structure, and arrestin post-translational modifications. We hypothesized that information about ligand and receptor structure is encoded in the conformation of the intracellular domains of an activated receptor and transferred allosterically to receptor-bound arrestin to dictate which of its many cellular functions it will perform. To test this hypothesis we developed a panel of arrestin3 intramolecular FlAsH BRET biosensors that allow detection of conformational shifts between the arrestin N-terminus and six positions within the protein. Measuring the effect of receptor activation on arrestin conformation generates an arrestin3 ‘conformational signature’ in a live cell, real time, multiwell plate format. Using a panel of structurally distinct angiotensin type 1A receptor (AT1AR) ligands, we show that GPCR-arrestin complex avidity correlates directly with the ligand-induced Δ Net BRET of an arrestin3 FlAsH-BRET sensor located within the arrestin3 C-terminal globular domain. We further hypothesized that perturbation of arrestin3 post-translational modifications that influence complex stability would similarly be reflected by loss of conformational shifts of arrestin characteristic of stable complex formation. Ubiquitination of arrestin3 at Lysines 11 and 12 is necessary to stabilize complexes with the AT1AR, but not the vasopressin type 2 receptors (V2R). We found that introduction of an arrestin3 K11/12R mutation, which changes the AT1AR-arrestin interaction from stable to transient, reduced the arrestin3 C-terminal FlAsH-BRET shift produced by AT1AR, but not by the V2R, whose trafficking is unaffected by the mutation. We further tested the impact of the K11/12R mutation on two previously unstudied receptors, the bradykinin type 2 receptor (B2R) and the type 1 parathyroid hormone receptor (PTH1R). Mutation resulted in loss arrestin3 FlAsH-BRET signal induced by B2R, but not PTH1R. Examination of arrestin trafficking by confocal microscopy demonstrated that the K11/12R mutation altered B2R, but not PTH1R, trafficking. We conclude that activation-induced changes in arrestin3 conformation, observable through intramolecular FlAsHBRET, reflect the impact of ligand structure and post translational-modification on its intracellular functions. Biophysical probes such as these, which predict the function of intracellular signaling proteins upon receptor activation, may have application in drug discovery efforts to identify “biased” ligands that tailor GPCR efficacy to elicit specific downstream signaling events

The risk for breast cancer is not evidently increased in women with hyperprolactinemia

The question has been raised whether hyperprolactinemia in humans is associated with an excess risk for breast cancer. We aimed to assess the risk of breast cancer in a previously defined large cohort of patients treated for idiopathic hyperprolactinemia or prolactinomas. Based on the pattern of drug prescriptions we identified 11,314 subjects in the PHARMO network with at least one dispensing of dopamine agonists between 1996 and 2006. Of these, 1,607 subjects were considered to have dopamine agonist—treated hyperprolactinemia based on the prescribing pattern. For the present analysis, we included only women (n = 1,342). Patients with breast cancer were identified by hospital discharge codes. Data on breast cancer incidence in the Netherlands were derived from the Dutch cancer registry. Standardized mortality ratio (SMR) was the measure of outcome to assess the association between hyperprolactinemia and breast cancer. The 1,342 patients accounted for a total of 6,576 person years. Eight patients with breast cancer during follow-up were identified. Indirect standardization with incidence proportions from the general Dutch population revealed a 7.47 expected cases. The calculated SMR for breast cancer risk in patients treated hyperprolactinemia was 1.07 (95% confidence interval 0.50–2.03). In conclusion, there is no clear evidence for increased breast cancer risk in female patients treated for either idiopathic hyperprolactinemia or prolactinomas. The uncertainty about the exact risk that is due to the relatively low number of breast cancer cases, should be overcome by pooling results in a future meta-analysis

The Role of β-Arrestin Proteins in Organization of Signaling and Regulation of the AT1 Angiotensin Receptor

AT1 angiotensin receptor plays important physiological and pathophysiological roles in the cardiovascular system. Renin-angiotensin system represents a target system for drugs acting at different levels. The main effects of ATR1 stimulation involve activation of Gq proteins and subsequent IP3, DAG, and calcium signaling. It has become evident in recent years that besides the well-known G protein pathways, AT1R also activates a parallel signaling pathway through β-arrestins. β-arrestins were originally described as proteins that desensitize G protein-coupled receptors, but they can also mediate receptor internalization and G protein-independent signaling. AT1R is one of the most studied receptors, which was used to unravel the newly recognized β-arrestin-mediated pathways. β-arrestin-mediated signaling has become one of the most studied topics in recent years in molecular pharmacology and the modulation of these pathways of the AT1R might offer new therapeutic opportunities in the near future. In this paper, we review the recent advances in the field of β-arrestin signaling of the AT1R, emphasizing its role in cardiovascular regulation and heart failure

Types of violence in TV series "Narcos" and its evaluation by the audience: expectations and need gratifications

Bakalaura darba Seriālā “Narcos” izmantotie vardarbības veidi un to novērtējums auditorijā mērķis ir identificēt seriālā atspoguļoto vardarbību un noskaidrot seriāla skatīšanās motivācijas un auditorijas vērtējumu par to, izpētot vai medija atspoguļotā vardarbība izraisa izmērāmus teorijā aprakstītus efektus. Teorētiskā daļā iekļauta lietojuma un apmierinājuma, kā arī gaidu-vērtību teorija un mediju pētnieku darbi par vardarbību medijos un to radītajiem efektiem. Empīriskajā daļā pielietota kontentanalīze un veiktas divas fokusa grupas diskusijas. Viens no galvenajiem pētījuma secinājumiem ir tāds, ka vardarbīga satura patērētāji pašvērtējumā neapzinās iespējamos negatīvos efektus no vardarbīga satura skatīšanāsThe aim of bachelor’s thesis Types of violence in TV series "Narcos" and its evaluation by the audience: expectations and need gratifications” is to identify violent scenes, which is shown in TV series, to study audience’s watching motivations and valuations about violence, and to find out, if reflected violence in TV series causes mesurable media effects. Theoretical framework of thesis includes uses and gratifications theory, expectancy- value theory and different scholars about violence in media. Content analysis and two focus group discussions are usesd in the empirical part of the study. One of the main research conclusion is that violent content viewers are not aware of potencial negative effects from violent content consumption