Functional modulation of PTH1R activation and signaling by RAMP2

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein–coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

G protein–coupled receptors(GPCRs) represent the largest class of membrane-bound proteins and are involved in a multitude of biological processes (1). They are characterized by a seven-transmembrane helix structure, which undergoes a characteristic rearrangement upon binding of agonists. Agonist binding to its cognate receptor induces conformational changes in the transmembrane helices, which are transmitted to the cytosolic face of the receptors and ultimately result in receptor activation, which represents the key step of signal transduction. The combination of crystallographic and cryogenic electron microscopy studies and the employment of optical biosensors to study the reorganization of the seven transmembrane domains has allowed a detailed understanding of the general mechanisms of GPCR activation (2–5).Earlier structural studies suggest that GPCRs undergo similar conformational changes upon activation, including, most prominently, an outward movement of the transmembrane helix 6 at the cytosolic face, thereby creating a pocket to which the G protein α-subunit can couple (5). More recent studies, however, have revealed that the exact type of changes may depend on the receptor class and the specific receptor (6–8). Class- and receptor-specific differences may also exist in the interaction of receptors not only with downstream G proteins and β-arrestins but also with accessory and modulatory proteins (9).Studies of the kinetic steps that govern the structural rearrangements which underlie receptor activation (10) showed that its speed might depend on the receptor class and the specific receptor. For example, when exposed to saturating agonist concentrations, most class A GPCRs switch into the active state within tens of milliseconds. The same process takes 1 to 2 ms for a class C GPCR and may take up to a second for class B receptors (11–15). Little is known whether the activation kinetics of GPCRs can be modulated by their cellular context and whether proteins other than the receptors themselves might play a role in shaping signaling kinetics and specificity.Here, we study the parathyroid hormone 1 receptor (PTH1R), a prototypical member of class B GPCRs characterized by a large N-terminal domain that binds a major part of their cognate peptide agonists (16, 17). Compared to class A GPCRs, PTH1R activation is relatively slow and occurs in a two-step process: The initial N-terminal binding step has a time constant of ∼140 ms, followed by an interaction of the ligand with the transmembrane core, which changes into its active conformation with a time constant of ∼1 s (11, 14). Pleiotropic in its downstream coupling, PTH1R signals primarily via G_s but can also couple to G_q (18), G_12/13 (19), and G_i (20) and interacts with and signals via β-arrestins (21, 22). The two endogenous agonists, parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), trigger PTH1R activation with similar kinetics and specificity for the various intracellular pathways (23–25). However, PTH can induce prolonged signaling from intracellular sites, while PTHrP signals exclusively from the cell surface (26).PTH1R has been reported to interact with modulatory proteins of the receptor-activity-modifying protein (RAMP) family (27–29). RAMPs constitute a family of single transmembrane helix proteins with three members: RAMP1, RAMP2, and RAMP3.It is controversial whether PTH1R interacts only or preferentially with RAMP2 (28) or all three RAMPs (28, 29). In RAMP2 knock-out mice, PTH1R function is deregulated, and placental dysfunction is observed (30), suggesting a major physiological role of the PTH1R/RAMP2 interaction. Yet, the molecular mechanisms of how RAMPs may modulate the activation dynamics of PTH1R and their signaling properties remain to be elucidated.To address these questions, we develop and employ biosensors for PTH1R activation and investigate an array of downstream signaling pathways to assess the effects of RAMPs on the activation dynamics and signaling properties of PTH1R in response to its two endogenous ligands, PTH and PTHrP. We observe that RAMP2 specifically interacts with PTH1R and modulates its activation kinetics as well as signaling dynamics in an agonist-dependent manner.     

Erythropoietin and intravenous iron therapy in postpartum anaemia

BACKGROUND: We assessed whether recombinant human erythropoietin (rhEPO) enhances a rise in haemoglobin concentration in postpartum anaemia compared to intravenous iron alone. DESIGN: Some 60 patients with haemoglobin values 相似文献   

Experiences of physical and sexual abuse and their implications for current health

OBJECTIVE: To estimate the prevalence of a history of physical and sexual abuse in adulthood among gynecological patients and the association with general and reproductive health. METHODS: A cross-sectional questionnaire study on abusive experiences of gynecologic outpatients in a tertiary hospital. The total sample size was 691. RESULTS: Of all women, 42.4% had experienced moderate or severe physical or sexual abuse as an adult. One hundred forty-seven (21.6%) women reported physical abuse, 84 (12.3%) sexual abuse, and 58 (8.5%) both. The abused and nonabused women did not differ in mean age, education, or parity. Sexually abused women and those who were both sexually and physically abused reported poor general health significantly more often (P=.005 and P=.001, respectively) than the nonabused. They also rated their sex life as significantly worse than the nonabused women (P=.002 and P=.012, respectively). Over half of abused women had experienced common physical complaints during the previous 12 months compared with one third of the nonabused (P<.001). Two thirds of both the abused and the nonabused women preferred that their gynecologist not ask directly about abuse. CONCLUSION: Abusive experiences were common in gynecologic outpatients. Women with abusive experiences had ill health and poor sexual life more often than the controls. In contrast to the results of previous studies, most of the women did not want to be asked about abuse by their gynecologist.     

Cycles of abuse nurtured by concealment: a clinical report

At present, health care staff do not seem to have sufficient knowledge about their patients' abusive experiences. The aim of the present study is to analyze and discuss what the implications might be for the encounter between patients and health care professionals, when experiences of abuse are concealed. The methodology of this article is varied: a personal narrative, medical records, sociological theoretical literature and empirical evidence. From the narrative we learn that concealment of abuse was devastating for the patient. She was "treated" in vain as a correct diagnosis was not made, while abuse by her father continued. Health care staff also violated her, which she told her therapist, but her protests were not acknowledged. Ten years of treatment thus made her even more sick. This case story focuses on the mechanisms which nurture concealment of a patient's history of abuse, such as structural and symbolic violence. We also suggest ways to break "cycles of abuse". Help the patient to stop concealing also means that she/he leaves a victim role, gets in charge of the situation and takes a first step towards empowerment. In this way, health care settings can become enabling and empowering environments.     

Cancer morbidity in nitrate fertilizer workers

Summary A cohort of 2,131 male nitrate fertilizer workers was evaluated for cancer morbidity from 1963 to 1986. No significant increase in total cancer, stomach cancer (5 actual vs 6.7 expected cases), or lung cancer (13 vs 13 expected) was found. On the other hand, 26 actual cases of prostate cancer were observed vs 16 expected cases (standardized morbidity ratio, SMR = 161; 95%, confidence interval, CI = 107–239). This risk increase however, was, not enhanced by applying at least a 10-year latency period. In a cohort of 1,148 male fertilizer workers who had never been exposed to nitrate, there was an increased incidence of lung cancer (SMR = 151,95% CI = 103–220) but not of stomach cancer or prostate cancer. There was no association between airborne nitrate exposure dose and total cancer, stomach cancer, lung cancer or prostate cancer, respectively.     

Haemostatic changes in pregnancy

Normal pregnancy is accompanied by changes in the coagulation and fibrinolytic systems. These include increases in a number of clotting factors (I, II, VII, VIII, IX and XII), a decrease in protein S levels and inhibition of fibrinolysis. As gestation progresses, there is also a significant fall in the activity of activated protein C, an important anticoagulant. While these physiological changes may be important for minimizing intrapartum blood loss, they entail an increased risk of thromboembolism during pregnancy and the post-partum period.     

Association of Average Telomere Length with Body-Mass Index and Vitamin D Status in Juvenile Population with Type 1 Diabetes

Background

Type 1 diabetes (T1D) is an autoimmune chronic disease where hyperglycemia, increased risk of oxidative stress, advanced glycation end-products and other genetic and environmental factors lead to T1D complications. Shorter telomeres are associated with hyperglycemic levels and lower serum vitamin D levels.

Methods

Average telomere length (ATL) in whole blood DNA samples was assessed with qPCR method in 53 Slovenian T1D children/adolescents (median age 8.7 years, 1:1.3 male/female ratio). Body mass index standard deviation score (BMI-SDS), glycated haemoglobin and serum level of vitamin D metabolite (25-(OH)-D3) and the age at the onset of T1D were collected from the available medical documentation.

Results

Results indicate shorter ATL in subjects with higher BMI-SDS when compared to those with longer ATL (0.455 ± 0.438, −0.63 ± 0.295; p=0.049). Subjects with higher BMI-SDS had lower serum vitamin D levels when compared to those with lower BMI-SDS (40.66 ± 3.07 vs. 52.86 ± 4.85 nmol/L; p=0.045). Vitamin D serum levels did not significantly differ between subjects with longer/shorter ATL.

Conclusion

T1D children/adolescents with shorter ATL tend to have higher BMI-SDS. Lower serum vitamin D levels were associated with higher BMI-SDS, while associations between vitamin D serum levels, age at the onset of T1D, glycated haemoglobin and ATL were not observed. Additional studies with more participants are required to clarify the role of the telomere dynamics in T1D aetiology and development of complications.     

Estimation of drug receptor occupancy when non-displaceable binding differs between brain regions – extending the simplified reference tissue model

AimThe simplified reference tissue model (SRTM) is used for estimation of receptor occupancy assuming that the non-displaceable binding in the reference region is identical to the brain regions of interest. The aim of this work was to extend the SRTM to also account for inter-regional differences in non-displaceable concentrations, and to investigate if this model allowed estimation of receptor occupancy using white matter as reference. It was also investigated if an apparent higher affinity in caudate compared with other brain regions, could be better explained by a difference in the extent of non-displaceable binding.MethodsThe analysis was based on a PET study in six healthy volunteers using the 5-HT_1B receptor radioligand [¹¹C]-AZ10419369. The radioligand was given intravenously as a tracer dose alone and following different oral doses of the 5-HT_1B receptor antagonist AZD3783. Non-linear mixed effects models were developed where differences between regions in non-specific concentrations were accounted for. The properties of the models were also evaluated by means of simulation studies.ResultsThe estimate (95% CI) of Ki_PL was 10.2 ng ml⁻¹ (5.4, 15) and 10.4 ng ml⁻¹ (8.1, 13.6) based on the extended SRTM with white matter as reference and based on the SRTM using cerebellum as reference, respectively. The estimate (95% CI) of Ki_PL for caudate relative to other brain regions was 55% (48, 62%).ConclusionsThe extended SRTM allows consideration of white matter as reference region when no suitable grey matter region exists. AZD3783 affinity appears to be higher in the caudate compared with other brain regions.     

Distribution of KIR genes in the Croatian population

The KIR locus with genes involved in immune processes is among the most polymorphic and structurally diverse human loci. KIR genes encode activating and inhibitory receptors that differ in specificity for HLA class I ligands and signaling potential. These receptors are expressed principally by natural killer (NK) cells and subpopulations of T cells. This study represents the first report of the distribution of KIR genes, KIR genotypes and KIR/HLA pairs in 121 unrelated healthy Croatian individuals. Twenty-three different genotypes were observed in the Croatian population and all 16 KIR genes known to date were found. The most frequent KIR genotype was the AA genotype. All individuals had at least one inhibitory KIR/HLA pair with the majority of individuals with three inhibitory KIR/HLA pairs. The most frequent KIR/HLA pair was the KIR2DL3/C1 group. Our results demonstrated the similarity of the Croatian population’s KIR repertoire with other Caucasian populations reported so far.