Role In Desensitization Research Articles

Interactions between AT1R and GRKs: the determinants for activation of signaling pathways involved in blood pressure regulation.

The success of Angiotensin II receptor blockers, specifically Angiotensin II type 1 receptor (AT1R) antagonists as antihypertensive drug emphasizes the involvement of AT1R in Essential hypertension. The structural insights and mutational studies of Ang II-AT1R have brought about the vision to design Ang II analogs that selectively activate the pathways with beneficial and cardioprotective effects such as cell survival and hinder the deleterious effects such as hypertrophy and cell death. AT1R belongs to G-protein coupled receptors and is regulated by G-protein coupled receptor kinases (GRKs) that either uncouples Gq protein for receptor desensitization or phosphorylate C-terminus to recruit β-arrestin for internalization of the receptor. The interaction of GRKs with ligand activated AT1R induces conformational changes and signal either Gq dependent or Gq independent pathways. These interactions might explain the complex regulatory mechanisms and offer promising ideas for hypertension therapeutics. This article reviews the functional role of AT1R, organization of GRK genes and regulation of AT1R by GRKs that play significant role in desensitization and internalization of the receptors.

Genetic Polymorphism of beta1-adrenergic Receptors and the Effect on the Clinical Efficacy of beta-adrenoblockers

Beta-adrenergic blockers are a valuable class of cardiovascular drugs and are widely used in the treatment of arterial hypertension (AH), coronary heart disease, chronic heart failure (CHF), cardiac arrhythmias, significantly improving the prognosis of patients. However, the clinical efficacy of betablockers is largely dependent on the genetic polymorphism of beta1-adrenergic receptors (ADRB1). The aim of the review was a systematic analysis of scientific data from pharmacogenetic studies on the role of beta1-adrenergic receptor polymorphism in the clinical efficacy of beta-blockers in the treatment of hypertension, chronic heart failure, and atrial fibrillation. The results of clinical trials and meta-analyzes were used. Of greatest importance is the genetic polymorphism of beta1-adrenergic receptors of two loci – Arg389Gly and Ser49Gly; the frequency of occurrence of variant and less functionally active alleles Gly389 and Gly49 in Europeans reaches 27% and 15%. The variant Gly389 allele has reduced functional activity and carriers have a weak response to the use of beta-blockers. In carriers of variant alleles Gly389 and Gly49 a reduced hypotensive effect on the use of beta-blockers was observed, and in studies of long-term efficacy, carriage of variant alleles was accompanied by an increase in the frequency and risk of unfavorable outcomes of hypertension. In pharmacogenetic studies, a reduced effect of the effect on myocardial remodeling in patients with CHF for beta-blockers in carriers of the variant Gly389 allele were confirmed. According to two meta-analyzes of trials on use of beta-blockers in patients with CHF, the frequency of increased left ventricle ejection fraction was significantly higher in carriers of the wild Arg389Arg gene type (risk ratio=1.83, p=0,001). In contrast, in atrial fibrillation, the frequency of rhythm control with beta-blockers was achieved better in the presence of the variant allele Gly389 with “loss of function”. Another polymorphic Gly49 allele plays a role in desensitization and down-regulation of beta1-receptor activity, although clinically this effect has been less obvious and contradictory. However, in studies, a more pronounced clinical effect of beta-blockers was observed in carriers of the wild genotype Ser49Ser, as well as in carriers of the haplotype Ser49Ser/Arg389Arg. Thus, genetic polymorphism ADRB1 may be another important predictor of the effectiveness of beta-blockers in clinical practice, which must be taken into account in the treatment of cardiovascular diseases.

Abstract 16292: Selective Platelet Protease-Activated-Receptor (PAR)1 and PAR4 Mediated Thrombin Desensitisation in the Elderly is Correlated With Inflammation and Chronic Thrombin Receptor Stimulation

Introduction: Platelet activation, by adenosine diphosphate (ADP) via P2Y 12 receptors and thrombin via PAR1 and PAR4, is a key therapeutic target in cardiovascular disease (CVD). The efficacy of antiplatelet agents diminishes in the elderly, but it is unknown whether these pathways change with aging. Hypothesis: Platelet activation pathways change with aging. Methods: Platelet activity was evaluated in young (20-30yrs), middle-aged (40-55yrs) and elderly (≥70yrs) healthy volunteers (n=174). Whole blood aggregometry and flow cytometry (P-selectin: α-granule release; CD63: dense granule release; PAC1 binding: activated GPIIb/IIIa) were performed under basal conditions and post ex vivo stimulation with ADP, thrombin, PAR1 agonist or PAR4 agonist. EC 50 and E max values were derived for each agonist. Receptor cleavage and quantification (P2Y 12 ; PAR1; PAR4; GPIbα) were assessed with flow cytometry. Thrombin generation (D-Dimer) and inflammation (interleukin [IL]-1β; tumour necrosis factor [TNF]-α) were assessed via ELISA. Results: The elderly had higher basal platelet activation markers (P-selectin, CD63, activated GPIIb/IIIa) than the young, with higher basal activity correlating with increasing IL-1β. P2Y 12 receptor density was higher in the elderly and associated with greater ADP-induced platelet aggregation and activation. Elderly subjects had less platelet activation in response to thrombin (higher EC 50 ), demonstrating hyporeactivity to selective stimulation of PAR1 or PAR4, more basal PAR1/PAR4 cleavage, and less inducible PAR1/PAR4 cleavage. This was associated with reduced thrombin binding receptor GPIbα and reduced secondary ADP contribution to thrombin-mediated activation. D-Dimer and TNF-α levels were elevated in the elderly, and inversely correlated with platelet thrombin sensitivity, implying a role of desensitization from chronic thrombin receptor stimulation. Conclusion: Aging is associated with increased basal platelet activation and hyperreactivity to ADP, but selective desensitization to thrombin. The latter appears mediated by chronic thrombin receptor stimulation and inflammation. Age-specific antiplatelet strategies may require selective targeting of these pathways to treat CVD in the elderly.

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR’s C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with “biased agonist” properties.

Managing highly sensitized renal transplant candidates in the era of kidney paired donation and the new kidney allocation system: Is there still a role for desensitization?

Kidney paired donation (KPD) and the new kidney allocation system (KAS) in the United States have led to improved transplantation rates for highly sensitized candidates. We aimed to assess the potential need for other approaches to improve the transplantation rate of highly sensitized candidates such as desensitization. Using the UNOS STAR file, we analyzed transplant rates in a prevalent active waiting-list cohort as of June 1, 2016, followed for 1year. The overall transplantation rate was 18.9% (11129/58769). However, only 9.7% (213/2204) of candidates with a calculated panel reactive antibody ≥99.9% received a transplant, and highly sensitized candidates were less likely to receive a living donor transplant. Among candidates with a CPRA≥99.5% (ie. 100%), only 2.5% of transplants were from living donors (13 total, 7 from KPD). Nearly 4years after KAS (6/30/2018), 1791 actively wait-listed candidates had a CPRA of ≥99.9% and 34.6% (620/1791) of these had ≥5years of waiting time. Thus, despite KPD and KAS, many sensitized candidates have not been transplanted even with prolonged waiting time. We conclude that candidates with a CPRA≥99.9% and sensitized candidates with an incompatible living donor and prolonged waiting time may benefit from desensitization to improve their ability to receive a transplant.

The Role of Desensitization in Kidney Paired Donation

Kidney paired donation (KPD), desensitization, and prioritization in the deceased donor kidney allocation system (KAS) are current options to facilitate kidney transplant in difficult-to-match, highly sensitized patients. KPD participation has increased while new desensitization tools are developed. We review these trends and discuss the combined role of desensitization and KPD. According to the National Kidney Registry (NKR), KPD utilizing desensitization decreased while the number of highly sensitized KPD transplants and deceased donor transplants increased over the past decade. This is likely because of large KPD registries and the national allocation of deceased donor kidneys that allow highly sensitized patients to find compatible matches without the need for desensitization. Highly sensitized patients with incompatible living donors should be entered into a KPD registry while simultaneously considered for deceased donor transplant. Those patients who do not find a compatible match within a year should be considered for desensitization.

Is There a Role for Desensitization in Intestinal Transplantation?

Human leukocyte antigen allosensitization prior to transplant can increase the risk of early graft loss and prolong waitlist times for intestinal transplant candidates. Desensitization offers a potential therapeutic option to reduce the quantity of preformed antibodies prior to organ allocation and facilitate transplantation with a more immunologically compatible donor allograft. However, there remains a paucity of data to guide the use of desensitization in the setting of intestinal transplantation. As a result, in this review we evaluate the existing literature supporting the role of desensitization therapy in intestinal transplant, describe our own experience with the implementation of a risk-stratified desensitization protocol, and finally explore barriers and unanswered questions that continue to limit the widespread adoption of desensitization as a management strategy for highly sensitized intestinal transplant candidates.

Oxaliplatin: Detection and Management of Hypersensitivity Reactions.

Oxaliplatin is used extensively for the treatment of gastrointestinal cancer and other malignancies, with increased frequency of use in recent years. Hypersensitivity reactions (HSRs) can pose a major problem in clinical practice because they can limit the use of oxaliplatin in the care of malignancies in which it has proven efficacy. Nurses play an integral role in the administration of oxaliplatin; therefore, they need to be well educated in the prevention, detection, and management of HSRs. This article reviews the symptoms of HSRs associated with oxaliplatin, the specific management of HSRs associated with oxaliplatin, the role of desensitization, and the potential use of skin testing to better identify patients at risk for HSR. This article reviews the literature related to the diagnosis, prevention, and management of HSRs associated with oxaliplatin and outlines nurses' role. Oxaliplatin HSRs can occur at any cycle, but patients are at highest risk after they have received six prior infusions of oxaliplatin.

Anti MICA Antibody (major histocompatibility complex class I related Chain A) Whether to Treat or Avoid?

Aims To study the role of MICA antibody in living renal transplantation in the absence of anti HLA antibodies. Methods A retrospective study of patients undergone living renal transplantation between 2000-2014 was performed. Recipients were classified in two groups, pre transplantation Anti MICA antibody positive group (n=17) and antibody negative comparison group (n=17). Patients with anti HLA antibodies were excluded and only isolated MICA positive patients were included in the study group. All recipients undergone tolerance protocol and post transplant T-regulatory cells, and were comparable in recipient age, donor age, donor relation, HLA and immunosuppression including induction. Results Patients with pre transplant MICA antibody positivity were associated with increased acute rejection as compared to comparison group (47% vs 11.7 %, p value= 0.02 ). Renal function in MICA positive group and comparison group were comparable over a mean follow up of 6.5 years (mean s.creatinine 1.58 vs 1.53 mg/dl ). Rate of chronic rejection was same in both groups (5.8% ). No patient or graft loss in either group over mean follow up of 6.5 years. Conclusion Isolated Anti MICA antibody positivity is uncommon event. Pre transplant anti MICA antibody positivity is associated with increased acute rejection rates. However chronic rejection rates and renal function are comparable in both groups. Thus our study emphasizes that MICA antibody positive patients may require more aggressive immunosuppression, role of desensitization has to be defined.

Tolerance Induction to Antituberculosis Drugs in a Patient With Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Overlap.

Tolerance induction and desensitization in Stevens–Johnson syndrome (SJS) or in toxic epidermal necrolysis (TEN) have been described as an absolute contraindication by some authors, but there are cases where there is no treatment alternative. Tuberculosis (TB) remains a leading cause of morbidity and mortality in developing countries and ranks alongside HIV as a leading cause of death worldwide. Severe drug reactions, such as SJS and TEN, occurring in these individuals are lifethreatening. Since alternative therapies for TB are limited, the role of desensitization and reintroduction becomes essential. We describe a case of tolerance induction to anti-TB drugs in a patient with SJS/TEN overlap syndrome using a specifically designed premedication, comedication, and desensitization protocol.

Anti MICA (Major Histocompatibility Complex Class I) Related Antibody, Whether to Treat or Avoid in Renal Transplantation?

Introduction Pre-transplantation anti-major histocompatibility complex class-I related chain A (MICA) sensitization is an uncommon event and its role in kidney graft evolution is not completely defined. • Even when kidney allografts are well matched for HLA as in living related transplant and antiHLA antibodies are not detected, graft rejection can still occur. • Anti MICA antibody is reportedly associated with poor transplant outcomes and a high risk of acute and chronic rejection in renal transplantation. Methods A retrospective study of patients undergone living renal transplantation between years 2000-2014 was performed. • Recipients were classified in two groups, pre-transplantation Anti MICA antibody positive group (n=17) and antibody negative comparison group (n=17). • Patients with anti HLA antibodies were excluded and only isolated MICA positive patients were included in the study group. • Both groups were comparable in recipient age, donor age, donor relation, HLA and immunosuppression. Results Patients with pre transplant MICA antibody positivity were associated with increased acute rejection rate as compared to comparison group (47% vs. 11.7 %, p value= 0.02). • Renal function in MICA positive group and comparison group were comparable over a mean follow up of 6.5 years (mean creatinine 1.58 vs. 1.53 mg/dl). Rate of chronic rejection was same in both groups (5.8%). • No patient loss or graft loss occurred in either group over mean follow up of 6.5 years. Conclusion IsolatedAnti MICA antibody positivity is uncommon event. • Pre transplant anti MICA antibody positivity is associated with increased acute rejection rates. However chronic rejection rates and renal function are comparable in both groups. • Thus our study emphasizes that MICA antibody positive patients may require more aggressive immunosuppression. Role of desensitization has to be defined.

GRP78 Impairs Production of Lipopolysaccharide-Induced Cytokines by Interaction with CD14.

The 78-kDa glucose-regulated protein (GRP78) is a stress-inducible chaperone that resides primarily in the endoplasmic reticulum. GRP78 has been described to be released at times of cellular stress and as having extracellular properties that are anti-inflammatory or favor the resolution of inflammation. In the current study, we confirmed that GRP78 impaired the production of lipopolysaccharide-induced pro-inflammatory cytokines in GRP78-treated bone-marrow-derived dendritic cells (DCs). To explore the underlying mechanism, first of all, GRP78 was checked to be bound to the plasma membrane. Interestingly, such binding promoted endocytosis of toll-like receptor (TLR) 4 and reduction in TLR4 on the plasma surface had a key role in desensitization of GRP78-treated DCs to lipopolysaccharide. Given that cluster of differentiation (CD)14 is a crucial regulator of TLR4 endocytosis, interaction of GRP78 with CD14 was investigated next. Data showed that GRP78 co-localized with CD14 on the plasma membrane and glutathione-S-transferase-GRP78 precipitated CD14. In CD14 knockout mice, down-regulation of tumor necrosis factor-α and reduction in TLR4 on the plasma surface were abrogated in GRP78-treated DCs. Overall, these data suggested that GRP78 mediates endocytosis of TLR4 by targeting CD14 to favor the resolution of inflammation.

Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs.

In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), β-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HT2C receptors, which engage Erk1/2 pathway via a β-arrestin-dependent mechanism, promotes MEK-dependent β-arrestin2 phosphorylation at Thr383, a necessary step for Erk recruitment to the receptor/β-arrestin complex and Erk activation. Likewise, Thr383 phosphorylation is involved in β-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as β2-adrenergic, FSH and CXCR4 receptors, but does not affect the β-arrestin-independent Erk1/2 activation by 5-HT4 receptor. Collectively, these data show that β-arrestin2 phosphorylation at Thr383 underlies β-arrestin-dependent Erk1/2 activation by GPCRs.

Clathrin-mediated endocytosis is responsible for the lysosomal degradation of dopamine D3 receptor

GRK2-/β-Arrestin- and PKA-/PKC-mediated desensitization, internalization, and degradation are three representative pathways for regulating G protein-coupled receptors (GPCRs). Compared with GRK2/β-arrestin-mediated ones, functional relationship among the aforementioned three regulatory processes mediated by PKA/PKC is less clear. Dopamine D3 receptor (D3R), a major target of currently available antipsychotic drugs, is a typical GPCR that selectively undergoes PKC-mediated regulation. In the present study, we examined PKC-mediated internalization of D3R in correlation with its roles in desensitization and degradation. Our results showed that the kinase activity of PKCβII and the 229th and 257th serine residues of D3R were required for PKC-mediated desensitization, internalization, and degradation of D3R. PMA treatment ubiquitinated D3R and induced its degradation through lysosomal pathway. Blockade of clathrin-mediated internalization inhibited PKC-mediated lysosomal degradation of D3R but did not affect its desensitization. These results suggested that PKC-mediated phosphorylation of D3R involved clathrin-mediated internalization, which was important for the lysosomal degradation of D3R.

Diagnosis and Management of Infusion-Related Hypersensitivity Reactions to Enzyme Replacement Therapy for Lysosomal Diseases: The Role of Desensitization

Diagnosis and Management of Infusion-Related Hypersensitivity Reactions to Enzyme Replacement Therapy for Lysosomal Diseases: The Role of Desensitization

Diagnosis and management of infusion-related hypersensitivity reactions to enzyme replacement therapy for lysosomal diseases: The role of desensitization

Diagnosis and management of infusion-related hypersensitivity reactions to enzyme replacement therapy for lysosomal diseases: The role of desensitization

Role of Phosphorylation Sites in Desensitization of µ-Opioid Receptor.

Phosphorylation of residues in the C-terminal tail of the µ-opioid receptor (MOPr) is thought to be a key step in desensitization and internalization. Phosphorylation of C-terminal S/T residues is required for internalization (Just et al., 2013), but its role in desensitization is unknown. This study examined the influence of C-terminal phosphorylation sites on rapid desensitization of MOPr. Wild-type MOPr, a 3S/T-A mutant (S363A, T370A, S375A) that maintains internalization, 6S/T-A (S363A, T370A, S375A, T376A, T379A, T383A) and 11S/T-A (all C-terminal S/T residues mutated) mutants not internalized by MOPr agonists were stably expressed in AtT20 cells. Perforated patch-clamp recordings of MOPr-mediated activation of G-protein-activated inwardly rectifying potassium channel (Kir3.X) (GIRK) conductance by submaximal concentrations of Met(5)-enkephalin (ME) and somatostatin (SST; coupling to native SST receptor [SSTR]) were used to examine desensitization induced by exposure to ME and morphine for 5 minutes at 37°C. The rates of ME- and morphine-induced desensitization did not correlate with phosphorylation using phosphorylation site-specific antibodies. ME-induced MOPr desensitization and resensitization did not differ from wild-type for 3S/T-A and 6S/T-A but was abolished in 11S/T-A. Morphine-induced desensitization was unaffected in all three mutants, as was heterologous desensitization of SSTR. Morphine-induced desensitization (but not ME) was reduced by protein kinase C inhibition in wild-type MOPr and abolished in the 11S/T-A mutant, as was heterologous desensitization. These findings establish that MOPr desensitization can occur independently of S/T phosphorylation and internalization; however, C-terminal phosphorylation is necessary for some forms of desensitization because mutation of all C-terminal sites (11S/T-A) abolishes desensitization induced by ME.

Conformational Changes Underlying Glutamate Receptor Gating

The gating mechanism of glutamate receptor ion channels involves conformational changes triggered by the binding of glutamate that open the ion channel, followed within milliseconds by nearly complete desensitization. In a collaboration with the Subramaniam Lab at NCI, cryo-EM structures of full length AMPA (GluA2) receptors, stabilized in the active state by allosteric modulators, reveal concerted conformational changes in the ligand binding domain that produce a vertical contraction accompanied by a corkscrew like rotation that leads to expansion of the linkers leading to the M3 helix bundle; in the desensitized state the ATD dimer pairs adopts multiple conformations with evidence for substantial reorganization of the ligand binding domain tetramer. By contrast, for the GluK2 kainate receptor the ATD dimer assembly remains intact in the desensitized state, while the LBD rearranges into a 4-fold symmetric structure in which the linkers leading to the M3 helix bundle adopt a contracted conformation that is however different from the closed state. Measurements of ATD tetramer association by analytical ultracentrifugation reveal a 1000-fold high higher affinity for GluK2 versus GluA2 that underlies the different behavior of the ATD in the desensitized state, movements of which however are secondary to and play no major role in desensitization.

Evaluation of the role of g protein-coupled receptor kinase 3 in desensitization of mouse odorant receptors in a Mammalian cell line and in olfactory sensory neurons.

Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated.

SAT0461 Infliximab Treatment in Children: Frequency of Infusion Reactions and Role of Desensitization

BackgroundInfliximab (IFL) is a monoclonal tumour necrosis factor alpha inhibiting antibody which is frequently used to treat children with refractory arthritis and uveitis. The most frequent and limiting adverse events...